Retinoblastoma gene mutations detected by whole exome sequencing of Merkel cell carcinoma.

Merkel cell carcinoma is a highly aggressive cutaneous neuroendocrine tumor that has been associated with Merkel cell polyomavirus in up to 80% of cases. Merkel cell polyomavirus is believed to influence pathogenesis, at least in part, through expression of the large T antigen, which includes a retinoblastoma protein-binding domain. However, there appears to be significant clinical and morphological overlap between polyomavirus-positive and polyomavirus-negative Merkel cell carcinoma cases. Although much of the recent focus of Merkel cell carcinoma pathogenesis has been on polyomavirus, the pathogenesis of polyomavirus-negative cases is still poorly understood. We hypothesized that there are underlying human somatic mutations that unify Merkel cell carcinoma pathogenesis across polyomavirus status, and to investigate we performed whole exome sequencing on five polyomavirus-positive cases and three polyomavirus-negative cases. We found that there were no significant differences in the overall number of single-nucleotide variations, copy number variations, insertion/deletions, and chromosomal rearrangements when comparing polyomavirus-positive to polyomavirus-negative cases. However, we did find that the retinoblastoma pathway genes harbored a high number of mutations in Merkel cell carcinoma. Furthermore, the retinoblastoma gene (RB1) was found to have nonsense truncating protein mutations in all three polyomavirus-negative cases; no such mutations were found in the polyomavirus-positive cases. In all eight cases, the retinoblastoma pathway dysregulation was confirmed by immunohistochemistry. Although polyomavirus-positive Merkel cell carcinoma is believed to undergo retinoblastoma dysregulation through viral large T antigen expression, our findings demonstrate that somatic mutations in polyomavirus-negative Merkel cell carcinoma lead to retinoblastoma dysregulation through an alternative pathway. This novel finding suggests that the retinoblastoma pathway dysregulation leads to an overlapping Merkel cell carcinoma phenotype and that oncogenesis occurs through either a polyomavirus-dependent (viral large T antigen expression) or polyomavirus-independent (host somatic mutation) mechanism.

Juvenile granulosa cell tumors: immunoreactivity for CD99 and Fli-1 and EWSR1 translocation status: a study of 11 cases.

The accurate diagnosis of a juvenile granulosa cell tumor (JGCT) can be challenging, as these neoplasms often exhibit morphologic features that overlap other ovarian neoplasms. In addition, the immunohistochemical profile exhibited by JGCT is fairly nonspecific and typically includes reactivity for CD99. Recently, we noted that JGCTs can show immunohistochemical expression of Fli-1, a transcription factor expressed by Ewing sarcoma, a neoplasm that is occasionally in the differential diagnosis of JGCT. We evaluated a series of JGCTs to determine whether Fli-1 is commonly expressed by these tumors and whether they demonstrate chromosomal arrangements in EWSR1. Cases diagnosed as JGCT (n=11) were immunohistochemically evaluated for expression of Fli-1 and CD99. Fluorescence in situ hybridization was performed on all cases to search for chromosomal rearrangements in EWSR1. All 11 of our cases exhibited positive immunohistochemical staining for Fli-1 and CD99. None of the cases demonstrated rearrangement in EWSR1 by fluorescence in situ hybridization. In cases of JGCT that cannot be reliably distinguished from Ewing sarcoma based on morphology and immunohistochemistry alone, fluorescence in situ hybridization testing for EWSR1 rearrangements seems to be a useful diagnostic adjunct for their separation.

TP53 pathway analysis in paediatric Burkitt lymphoma reveals increased MDM4 expression as the only TP53 pathway abnormality detected in a subset of cases.

The TP53 (p53) pathway can be inhibited by TP53 mutation or deletion or by MDM2 overexpression. Both occur in Burkitt lymphoma (BL), but many cases lack either abnormality. Expression patterns of the TP53 inhibitor MDM4 have not been reported in BL, and increased MDM4 could deregulate the TP53 pathway in cases without TP53 or MDM2 abnormalities. We investigated TP53 pathway disruption in paediatric BL patient samples (n = 30) by studying MDM4, MDM2, and CDKN1A (p21) protein and mRNA expression; TP53 mutations; TP53 protein expression; and gene copy number abnormalities. MDM4 protein was expressed in 30/30 tumours, and MDM2 protein was weakly expressed in 7/30 (23%). All cases were negative for CDKN1A protein, and CDKN1A mRNA levels were decreased. TP53 mutations were detected in 5/28 (18%) cases and confirmed by sequencing. TP53 protein was expressed in 15/30 (50%) cases, including 7/8 with TP53 genetic alterations. MDM2 protein and mRNA expression levels did not correlate with lack of TP53 genetic changes or TP53 protein expression; however, there was an inverse relationship between detectable TP53 protein expression and MDM4 copy number gains and mRNA expression. The TP53 pathway is deregulated in paediatric BL cases, and increased MDM4 expression may be the primary mechanism in some cases.

Collateral deletion of the mitochondrial AAA+ ATPase ATAD1 sensitizes cancer cells to proteasome dysfunction.

The tumor suppressor gene PTEN is the second most commonly deleted gene in cancer. Such deletions often include portions of the chromosome 10q23 locus beyond the bounds of PTEN itself, which frequently disrupts adjacent genes. Coincidental loss of PTEN-adjacent genes might impose vulnerabilities that could either affect patient outcome basally or be exploited therapeutically. Here, we describe how the loss of ATAD1, which is adjacent to and frequently co-deleted with PTEN, predisposes cancer cells to apoptosis triggered by proteasome dysfunction and correlates with improved survival in cancer patients. ATAD1 directly and specifically extracts the pro-apoptotic protein BIM from mitochondria to inactivate it. Cultured cells and mouse xenografts lacking ATAD1 are hypersensitive to clinically used proteasome inhibitors, which activate BIM and trigger apoptosis. This work furthers our understanding of mitochondrial protein homeostasis and could lead to new therapeutic options for the hundreds of thousands of cancer patients who have tumors with chromosome 10q23 deletion.

Cancer cells have often lost genetic sequences that control when and how cell division takes place. Deleting these genes, however, is not an exact art, and neighboring sequences regularly get removed in the process. For example, the loss of the tumor suppressor gene PTEN, the second most deleted gene in cancer, frequently involves the removal of the nearby ATAD1 gene. While hundreds of thousands of human tumors completely lack ATAD1, individuals born without a functional version of this gene do not survive past early childhood. How can tumor cells cope without ATAD1 ­ and could these coping strategies become the target for new therapies? Winter et al. aimed to answer these questions by examining a variety of cancer cells lacking ATAD1 in the laboratory. Under normal circumstances, the enzyme that this gene codes for sits at the surface of mitochondria, the cellular compartments essential for energy production. There, it extracts any faulty, defective proteins that may otherwise cause havoc and endanger mitochondrial health. Experiments revealed that without ATAD1, cancer cells started to rely more heavily on an alternative mechanism to remove harmful proteins: the process centers on MARCH5, an enzyme which tags molecules that require removal so the cell can recycle them. Drugs that block the pathway involving MARCH5 already exist, but they have so far been employed to treat other types of tumors. Winter et al. showed that using these compounds led to the death of cancerous ATAD1-deficient cells, including in human tumors grown in mice. Overall, this work demonstrates that cancer cells which have lost ATAD1 become more vulnerable to disruptions in the protein removal pathway mediated by MARCH5, including via already existing drugs. If confirmed by further translational work, these findings could have important clinical impact given how frequently PTEN and ATAD1 are lost together in cancer.

ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma.

Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is typically initiated by inactivation of the von Hippel Lindau (VHL) gene, which results in the constitutive activation of the hypoxia inducible factors, HIF-1α and HIF-2α. Using a high throughput screen, we identify novel compounds that decrease HIF-1/2α levels and induce ferroptosis by targeting Iron Sulfur Cluster Assembly 2 (ISCA2), a component of the late mitochondrial Iron Sulfur Cluster (L-ISC) assembly complex. ISCA2 inhibition either pharmacologically or using siRNA decreases HIF-2α protein levels by blocking iron-responsive element (IRE)-dependent translation, and at higher concentrations, also decreases HIF-1α translation through unknown mechanisms. Additionally, ISCA2 inhibition triggers the iron starvation response, resulting in iron/metals overload and death via ferroptosis. ISCA2 levels are decreased in ccRCC compared to normal kidney, and decreased ISCA2 levels are associated with pVHL loss and with sensitivity to ferroptosis induced by ISCA2 inhibition. Strikingly, pharmacological inhibition of ISCA2 using an orally available ISCA2 inhibitor significantly reduced ccRCC xenograft growth in vivo, decreased HIF-α levels and increased lipid peroxidation, suggesting increased ferroptosis in vivo. Thus, the targeting of ISCA2 may be a promising therapeutic strategy to inhibit HIF-1/2α and to induce ferroptosis in pVHL deficient cells.

KIT mutations in ocular melanoma: frequency and anatomic distribution.

KIT mutations are known to occur in ~15% of chronic sun damaged cutaneous, mucosal, and acral melanomas. Melanomas with demonstrated activating mutations in KIT or platelet-derived growth factor receptor A (PDGFRA) may benefit from treatment with tyrosine kinase inhibitors. Currently, the limited data regarding KIT mutational status in ocular melanoma suggest that activating mutations are extremely rare. PDGFRA mutational status in ocular melanoma has not been determined. Seventy-five ocular melanomas (53 choroidal, 6 iris, 11 ciliary body, and 5 conjuctival) were selected from the files of the Department of Ophthalmology. High-resolution melting curve analysis and sequencing were performed to detect mutations in KIT exons 9, 11, 13, and 17 and PDGFRA exons 12 and 18. Results of mutational analysis were correlated with anatomical site and KIT (CD117) immunohistochemistry. Eight of 75 (11%) ocular melanomas contained mutations in either the KIT or PDGFRA gene. Five of 53 (9%) choroidal melanomas were associated with mutations (KIT exon 11=3; KIT exon 17=1; PDGFRA intron 18=1). Two of six (33%) iris melanomas and a single (9%) ciliary body melanoma harbored KIT exon 11 mutations. No mutations were identified in conjunctival melanomas. The distribution of KIT and PDGFRA mutations by ocular melanoma anatomical site did not reach statistical significance (P=0.393) CD117 positivity was not predictive of KIT mutational status as only 6 of 58 (10%) CD177-positive tumors harbored KIT mutations. In addition, a KIT exon 17 mutation was identified in one CD117-negative tumor. KIT and PDGFRA mutations do occur in ocular melanomas at a frequency (11%) that is similar to acral and mucosal melanomas. Limited correlation of CD117 positivity with mutational status suggests that all ocular melanomas should undergo mutational analysis to determine if imatinib therapy is appropriate.

The proteomic signature of NPM/ALK reveals deregulation of multiple cellular pathways.

Constitutive expression of the chimeric NPM/ALK fusion protein encoded by the t(2;5)(p32;q35) is a key oncogenic event in the pathogenesis of most anaplastic large cell lymphomas (ALCLs). The proteomic network alterations produced by this aberration remain largely uncharacterized. Using a mass spectrometry (MS)-driven approach to identify changes in protein expression caused by the NPM/ALK fusion, we identified diverse NPM/ALK-induced changes affecting cell proliferation, ribosome synthesis, survival, apoptosis evasion, angiogenesis, and cytoarchitectural organization. MS-based findings were confirmed using Western blotting and/or immunostaining of NPM/ALK-transfected cells and ALK-deregulated lymphomas. A subset of the proteins distinguished NPM/ALK-positive ALCLs from NPM/ALK-negative ALCLs and Hodgkin lymphoma. The multiple NPM/ALK-deregulated pathways identified by MS analysis also predicted novel biologic effects of NPM/ALK expression. In this regard, we showed loss of cell adhesion as a consequence of NPM/ALK expression in a kinase-dependent manner, and sensitivity of NPM/ALK-positive ALCLs to inhibition of the RAS, p42/44ERK, and FRAP/mTOR signaling pathways. These findings reveal that the NPM/ALK alteration affects diverse cellular pathways, and provide novel insights into NPM/ALK-positive ALCL pathobiology. Our studies carry important implications for the use of MS-driven approaches for the elucidation of neoplastic pathobiology, the identification of novel diagnostic biomarkers, and pathogenetically relevant therapeutic targets.

Macrophage HIF-1α Is an Independent Prognostic Indicator in Kidney Cancer.

PURPOSE: Clear cell renal cell carcinoma (ccRCC) is frequently associated with inactivation of the von Hippel-Lindau tumor suppressor, resulting in activation of HIF-1α and HIF-2α. The current paradigm, established using mechanistic cell-based studies, supports a tumor promoting role for HIF-2α, and a tumor suppressor role for HIF-1α. However, few studies have comprehensively examined the clinical relevance of this paradigm. Furthermore, the hypoxia-associated factor (HAF), which regulates the HIFs, has not been comprehensively evaluated in ccRCC. EXPERIMENTAL DESIGN: To assess the involvement of HAF/HIFs in ccRCC, we analyzed their relationship to tumor grade/stage/outcome using tissue from 380 patients, and validated these associations using tissue from 72 additional patients and a further 57 patients treated with antiangiogenic therapy for associations with response. Further characterization was performed using single-cell mRNA sequencing (scRNA-seq), RNA-in situ hybridization (RNA-ISH), and IHC. RESULTS: HIF-1α was primarily expressed in tumor-associated macrophages (TAMs), whereas HIF-2α and HAF were expressed primarily in tumor cells. TAM-associated HIF-1α was significantly associated with high tumor grade and increased metastasis and was independently associated with decreased overall survival. Furthermore, elevated TAM HIF-1α was significantly associated with resistance to antiangiogenic therapy. In contrast, high HAF or HIF-2α were associated with low grade, decreased metastasis, and increased overall survival. scRNA-seq, RNA-ISH, and Western blotting confirmed the expression of HIF-1α in M2-polarized CD163-expressing TAMs. CONCLUSIONS: These findings highlight a potential role of TAM HIF-1α in ccRCC progression and support the reevaluation of HIF-1α as a therapeutic target and marker of disease progression.

Microsatellite instability and survival in rectal cancer.

OBJECTIVE: High levels of microsatellite instability (MSI-H) have been associated in many studies with improved prognosis in colon cancer. Very few studies have evaluated the effect of MSI-H on rectal cancer survival. We assessed MSI-H and other genetic and epigenetic changes on survival of 990 individuals diagnosed with first primary rectal cancer. METHODS: MSI was assessed primarily by instability in the mononucleotide repeat BAT-26. The BRAF V600E mutation was assessed by TaqMan assay. The CpG island methylator phenotype (CIMP) was determined by methylation-specific PCR of CpG islands in MLH1, methylated in tumors (MINT)1, (MINT)2, (MINT)31 and CDKN2A. KRAS2 codons 12 and 13 mutations, and TP53 mutations in exons 5-8 were determined by sequencing. RESULTS: Multivariate analysis revealed that MSI-H (HRR 2.47, 95% CI 1.13-5.40) and KRAS2 mutations (HRR 1.37, 95% CI 1.04-1.81) were associated with a significantly higher risk of dying of rectal cancer. Only one of 22 MSI-H tumors showed a BRAF V600E mutation. Of 15 MSI-H rectal cancers evaluated for methylation, two exhibited MLH1 methylation and four exhibited CIMP. CONCLUSION: The genetic and epigenetic characteristics of MSI-H rectal cancers suggest that they are enriched for Lynch-associated tumors; adverse prognosis associated with MSI-H in these tumors may reflect the relatively high frequency of Lynch-associated cancers and/or the effect of radiation or chemotherapy on Lynch-associated rectal cancers or MSI tumors in general.

Silencing of HIF-1alpha by RNA interference in human glioma cells in vitro and in vivo.

Higher-grade gliomas are distinguished by increased vascular endothelial cell proliferation and peritumoral edema. These are thought to be instigated by vascular endothelial growth factor, which in turn is regulated by cellular oxygen tension. Hypoxia inducible factor-1alpha (HIF-1alpha) is a main responder to intracellular hypoxia and is overexpressed in many human cancers, including gliomas. Here we present methods for investigating the role of HIF-1alpha in glioma growth in vivo and in vitro using RNA interference in U251, U87, and U373 glioma cells.

Diffuse large B-cell lymphoma with coexpression of CD3 in a pediatric patient: a case report, review of the literature, and tissue microarray study.

The aberrant expression of T-cell antigens on B-cell-derived non-Hodgkin lymphomas has been described. However, the expression of the lineage-specific T-cell antigen, CD3, in hematologic malignancies is exceedingly rare and to the best of our knowledge has not been reported in pediatric patients. Here we describe the first case of a CD3+ diffuse large B-cell lymphoma in a 9-year-old male patient that is well documented by immunohistochemistry. In addition, results of a tissue microarray study composed of B-cell-derived non-Hodgkin lymphomas (n=77) and reactive lymphoid hyperplasia (n=13) dual stained for PAX5/CD3 are also reported.

Characterization of childhood precursor T-lymphoblastic lymphoma by immunophenotyping and fluorescent in situ hybridization: a report from the Children's Oncology Group.

BACKGROUND: T-lymphoblastic lymphoma (T-LBL) accounts for 25-30% of childhood non-Hodgkin's lymphoma and is closely related to T-lymphoblastic leukemia (T-ALL). Recently, we demonstrated distinct differences in gene expression between childhood T-LBL and T-ALL, but molecular pathogenesis and relevant protein expression patterns in T-LBL remain poorly understood. PROCEDURE: Children with T-LBL with disseminated disease were registered and treated on COG protocol 5971. Paraffin-embedded tumor tissue was obtained at diagnosis for immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) studies. We determined the pattern and intensity of staining for c-Myc, Skp2, Mib-1, p53, TCL-1, bcl-2, and bcl-6 proteins by IHC and c-Myc, p53, bcl-2, bcl-6, and TCR alpha/delta molecular alterations by FISH in 22 pediatric T-LBL cases. RESULTS: The majority of T-LBL samples expressed Mib-1 (59%) and c-Myc (77%) proteins in greater than 50% of the cells, but Skp2 (14%), p53 (14%), and bcl-2 (23%) expression was less common. FISH studies demonstrated 18% gains and 10% losses in c-Myc, 16% gains in p53, 12% gains and 6% losses in bcl-2, and 6% gains and 19% losses in bcl-6 with little direct correlation between the IHC and FISH studies. CONCLUSIONS: Childhood T-LBL is a highly proliferative tumor associated with enhanced expression of c-Myc protein, but without detectable c-Myc molecular alterations. FISH studies did not identify consistent etiologies of molecular dysregulation, and future studies with other molecular approaches may be required to elucidate the molecular pathogenesis of childhood T-LBL.

Global proteome profiling of NPM/ALK-positive anaplastic large cell lymphoma.

OBJECTIVE: Constitutive overexpression of nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) is a key oncogenic event in anaplastic large cell lymphomas (ALCL) that carry the t(2;5)(p23;q35) translocation. Global proteomic analysis of NPM/ALK-positive ALCL would improve understanding of the disease pathogenesis and yield new candidate targets for novel treatment and diagnostic strategies. MATERIALS AND METHODS: To comprehensively determine the inventory of proteins from NPM/ALK-positive ALCL SUDHL-1 cells, the membrane, cytoplasm, and nuclear subcellular fractions were resolved by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The MS spectra were interpreted using SEQUEST to search the electronic UniProt protein database, and analyzed by ProteinProphet and INTERACT. RESULTS: A total of 623 proteins consisting of 210 membrane, 229 cytoplasm, and 184 nuclear proteins were identified with a

IDH1R132H is intrinsically tumor-suppressive but functionally attenuated by the glutamate-rich cerebral environment.

Recurrent heterozygous mutation of isocitrate dehydrogenase 1 gene (IDH1), predominantly resulting in histidine substitution at arginine 132, was first identified in glioma. The biological significance of IDH1R132H, however, has been controversial, and its prevalent association with glioma remains enigmatic. Although recent studies indicate that IDH1R132H is nonessential to tumor growth or even anti-tumor growth, whether IDH1R132H initiates gliomagenesis remains obscure. In this study, we report that IDH1R132H is intrinsically tumor-suppressive but the activity can be attenuated by glutamate-the cerebral neurotransmitter. We observed that IDH1R132H was highly suppressive of subcutaneous tumor growth driven by platelet-derived growth factor B (PDGFB), but IDH1R132H tumor growth and glioma penetrance were virtually indistinguishable from those of IDH1-wildtype tumors in orthotopic models. In vitro, addition of glutamate compromised IDH1R132H inhibition of neurosphere genesis, indicating glutamate promotion of oncogenic dominance. Furthermore, we observed that IDH1R132H expression was markedly decreased in tumors but became more permissible upon the deletion of tumor-suppressor gene Cdkn2a. To provide direct evidence for the opposing effect of IDH1R132H on PDGFB-driven glioma development, we explored tandem expression of the two molecules from a single transcript to preclude selection against IDH1R132H expression. Our results demonstrate that when juxtaposed with oncogenic PDGFB, IDH1R132H overrides the oncogenic activity and obliterates neurosphere genesis and gliomagenesis even in the glutamate-rich microenvironment. We propose therefore that IDH1R132H is intrinsically suppressive of glioma initiation and growth but such tumor-suppressive activity is compromised by the glutamate-rich cerebral cortex, thereby offering a unifying hypothesis for the perplexing role of IDH1R132H in glioma initiation and growth.

Mutant IDH1 Promotes Glioma Formation In Vivo.

Isocitrate dehydrogenase 1 (IDH1) is the most commonly mutated gene in grade II-III glioma and secondary glioblastoma (GBM). A causal role for IDH1R132H in gliomagenesis has been proposed, but functional validation in vivo has not been demonstrated. In this study, we assessed the role of IDH1R132H in glioma development in the context of clinically relevant cooperating genetic alterations in vitro and in vivo. Immortal astrocytes expressing IDH1R132H exhibited elevated (R)-2-hydroxyglutarate levels, reduced NADPH, increased proliferation, and anchorage-independent growth. Although not sufficient on its own, IDH1R132H cooperated with PDGFA and loss of Cdkn2a, Atrx, and Pten to promote glioma development in vivo. These tumors resembled proneural human mutant IDH1 GBM genetically, histologically, and functionally. Our findings support the hypothesis that IDH1R132H promotes glioma development. This model enhances our understanding of the biology of IDH1R132H-driven gliomas and facilitates testing of therapeutic strategies designed to combat this deadly disease.

Comprehensive identification of proteins in Hodgkin lymphoma-derived Reed-Sternberg cells by LC-MS/MS.

Mass spectrometry-based proteomics in conjunction with liquid chromatography and bioinformatics analysis provides a highly sensitive and high-throughput approach for the identification of proteins. Hodgkin lymphoma is a form of malignant lymphoma characterized by the proliferation of Reed-Sternberg cells and background reactive lymphocytes. Comprehensive analysis of proteins expressed and released by Reed-Sternberg cells would assist in the discovery of potential biomarkers and improve our understanding of its pathogenesis. The subcellular proteome of the three cellular compartments from L428 and KMH2 Hodgkin lymphoma-derived cell lines were fractionated, and analyzed by reverse-phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Additionally, proteins released by Hodgkin lymphoma-derived L428 cells were extracted from serum-free culture media and analyzed. Peptide spectra were analyzed using TurboSEQUEST against the UniProt protein database (5.26.05; 188 712 entries). A subset of the identified proteins was validated by Western blot analysis, immunofluorescence microscopy and immunohistochemistry. A total of 1945 proteins were identified with 785 from the cytosolic fraction, 305 from the membrane fraction, 441 from the nuclear fraction and 414 released proteins using a minimum of two peptide identifications per protein and an error rate of <5.0%. Identification of proteins from diverse functional groups reflected the functional complexity of the Reed-Sternberg proteome. Proteins with previously reported oncogenic function in other cancers and from signaling pathways implicated in Hodgkin lymphoma were identified. Selected proteins without previously demonstrated expression in Hodgkin lymphoma were validated by Western blot analysis (B-RAF, Erb-B3), immunofluorescence microscopy (Axin1, Tenascin-X, Mucin-2) and immunohistochemistry using a tissue microarray (BRAF, PIM1). This study represents the first comprehensive inventory of proteins expressed by Reed-Sternberg cells of Hodgkin lymphoma and demonstrates the utility of combining cellular subfractionation, protein precipitation, tandem mass spectrometry and bioinformatics analysis for comprehensive identification of proteins that may represent potential biomarkers of the disease.

Comparison of Dual-ISH (DISH) With Fluorescence In Situ Hybridization (FISH) and Correlation With Immunohistochemical Findings for HER2/Neu Status in Breast Carcinoma.

INTRODUCTION: The most widely used methods for determination of HER2/neu status in breast carcinoma are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Both techniques are associated with technical and interpretive difficulties. Alternative methods exist including quantitative PCR and the newly developed chromogenic dual in situ hybridization (DISH). METHODS: We evaluated HER2 DISH as an alternative to FISH and report our findings from 101 cases. In addition, we correlated HER2 DISH and FISH results with HercepTest and 4B5 immunohistochemistry. RESULTS: Eight cases failed FISH analysis and none failed DISH analysis. A 95% (88/93) concordance was found between DISH and FISH for all cases in the series. When only 2+ IHC cases were evaluated, the concordance was 94% for DISH and FISH. Using the 2013 ASCO/CAP recommendations, none of the tested cases were equivocal by FISH or DISH despite 66% of cases being 2+ by HercepTest and 32% by the 4B5 antibody. COMMENT: Our study, which utilizes a majority of IHC equivocal cases, demonstrates that HER2 FISH and DISH are concordant methodologies. HER2 DISH is therefore an acceptable alternative to FISH.

SCF-KIT signaling induces endothelin-3 synthesis and secretion: Thereby activates and regulates endothelin-B-receptor for generating temporally- and spatially-precise nitric oxide to modulate SCF- and or KIT-expressing cell functions.

We demonstrate that SCF-KIT signaling induces synthesis and secretion of endothelin-3 (ET3) in human umbilical vein endothelial cells and melanoma cells in vitro, gastrointestinal stromal tumors, human sun-exposed skin, and myenteric plexus of human colon post-fasting in vivo. This is the first report of a physiological mechanism of ET3 induction. Integrating our finding with supporting data from literature leads us to discover a previously unreported pathway of nitric oxide (NO) generation derived from physiological endothelial NO synthase (eNOS) or neuronal NOS (nNOS) activation (referred to as the KIT-ET3-NO pathway). It involves: (1) SCF-expressing cells communicate with neighboring KIT-expressing cells directly or indirectly (cleaved soluble SCF). (2) SCF-KIT signaling induces timely local ET3 synthesis and secretion. (3) ET3 binds to ETBR on both sides of intercellular space. (4) ET3-binding-initiated-ETBR activation increases cytosolic Ca2+, activates cell-specific eNOS or nNOS. (5) Temporally- and spatially-precise NO generation. NO diffuses into neighboring cells, thus acts in both SCF- and KIT-expressing cells. (6) NO modulates diverse cell-specific functions by NO/cGMP pathway, controlling transcriptional factors, or other mechanisms. We demonstrate the critical physiological role of the KIT-ET3-NO pathway in fulfilling high demand (exceeding basal level) of endothelium-dependent NO generation for coping with atherosclerosis, pregnancy, and aging. The KIT-ET3-NO pathway most likely also play critical roles in other cell functions that involve dual requirement of SCF-KIT signaling and NO. New strategies (e.g. enhancing the KIT-ET3-NO pathway) to harness the benefit of endogenous eNOS and nNOS activation and precise NO generation for correcting pathophysiology and restoring functions warrant investigation.

A comparison of immunohistochemical stain quality in conventional and rapid microwave processed tissues.

Same-day turnaround of pathology specimens is desirable in this era of managed care, and rapid microwave tissue processing produces histologic features of a quality equivalent to overnight processing. We studied whether microwave-assisted rapid tissue processing adversely affects the quality of immunohistochemical staining. We selected 30 specimens (20 neoplastic and 10 nonneoplastic) from our routine surgical pathology workload. Paired large tissue blocks were made from each specimen type, one for microwave-assisted rapid processing and one for conventional processing. Two microarrays of 60 punches each were made from the donor blocks. The microarray blocks were examined for intensity and extent of staining by 44 commonly used antibodies. Slides were reviewed independently by 2 pathologists blinded to the type of processing used. In 5,280 tissue punches examined, we found a high degree of concordance in quality, as measured by intensity and extent of immunohistochemical staining, between microwave and routinely processed tissues. Our study demonstrates that quality of immunohistochemical staining is similar between rapid microwave and conventional processing. The potential need for immunohistochemical analysis is not a contraindication for microwave-assisted rapid tissue processing.