Diffuse Cytoplasmic Pattern Research Articles

P0304 SARS-COV-2 in Colorectal Cancer of IBD Patients

Abstract Background It has been hypothesized that inflammation related to SARS-CoV-2 infection can affect cancer cell proliferation, without having a direct oncogenic effect. SARS-CoV-2 infection is associated with the ACE2 receptor. ACE-2 over-expression has been found in many kinds of malignancies, including rectum adenocarcinoma (ADC) and in IBD patients. IBD-associated CRC (colorectal cancer) arises from a specific carcinogenic pathway involving chronic inflammation which is distinct from the traditional pathways. IBD patients characteristically had right colon cancer and a higher incidence with respect to the general population, which had declined over time. Methods We observed a sudden increase in CRC incidence. Between June 2022 and May 2024, 6 of our patients presented with ano-rectal cancer (4 in the lower rectum and two in the anal canal). Biopsies were reviewed as usual and an extra study searching for coronavirus protein was also performed. Immunohistochemical analysis was performed using OMNIS Automated System (AGILENT), and monoclonal antibody anti-SARS-COV-2 clone 1A9 from Gene Tex. Inc (USA) at 1:100. All patients had signed consent. Results They were male, ages 38-62, one smoked and one was an ex-smoker, duration of the disease 3-31 years, two were overweight. 3 Crohn’s Disease (CD) and 3 Ulcerative Colitis (UC). CD: A3L1B2, A2L2B1 and A1L3B3p. UC: 2 extensive and one distal. All on different treatments: adalimumab and azathioprine, azathioprine and steroids, filgotinib, two were on vedolizumab and one was only on oral mesalamine (never biologics). 5/6 were in endoscopical remission. They were diagnosed of ADC of distal rectum and two of perianal ADC. Four of them were mucinous ADC (including the two perianal ones), one conventional low grade infiltrant ADC (pT1) in distal rectum-anus, and the sixth with rectal intramucous ADC. 100% had received COVID vaccines (variable type) and had had COVID infections. Biopsies: all frequent viruses were negative, whereas coronavirus was found in all cases, with a diffuse cytoplasmic and granular pattern. See figures 1 and 2. Conclusion The incidence of CRC in our patients in the past two years is relevant. The type of tumour and its locations also calls our attention, as well as the fact that all tumours have SARS-COV-2 protein abundance. Whether an epiphenomenom, part of the cause or a trigger for CRC of this specific type, will have to be dilucidated in prospective studies. We do not know if coronavirus stays longer in the bowel mucosae of our IBD patients (long covid) or maybe just in a subpopulation. It is of clinical relevance no matter whether the coronavirus is a mere spectator or an actor. Many questions arise with just these six patients, which we hope to answer in the future.

SIRT4 is associated with microvascular infiltration, immune cell infiltration, and epithelial mesenchymal transition in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. In the present study, we evaluated SIRT4 expression levels in HCC specimens and investigated the relationships between SIRT4 expression levels, clinicopathological factors, and microvascular infiltration (MVI) in HCC. The expression levels of SIRT4 in 108 HCC specimens were examined by immunohistochemical staining. MVI in HCC specimens was divided into three subtypes: M0, M1, and M2. Comprehensive bioinformatics analysis was carried out to demonstrate SIRT4's biological functions and expression-related prognostic value. The diffuse cytoplasmic expression pattern of SIRT4 was observed in all adjacent nonneoplastic liver tissues. The levels of SIRT4 were higher in HCC than in any other type of cancer and normal tissues. In addition, the expression levels of SIRT4 were significantly decreased in HCC tissues when MVI was M1 or M2 (P=0.003) but were not related to the overall clinical outcome. To explain MVI regulated by SIRT4, we also found that SIRT4 expression correlated with epithelial-mesenchymal transition (EMT) markers and CD4+ T/NK cells and downregulated cancer-associated fibroblast cells. Also, there was a significant relationship between MVI and degree of cell differentiation (P=0.003), tumor size (P<0.001), alpha fetoprotein (AFP) (P=0.001), alanine aminotransferase (ALT) (P=0.024), and γ-glutamyl transferase (γ-GT) (P=0.024). However, SIRT4 was not an independent prognostic marker of HCC. Our results demonstrated an association between SIRT4 expression levels, MVI, immune cell infiltration, and potential biological functions, including EMT in the progression of HCC.

Immunolocalization of the AT-1R Ang II Receptor in Human Kidney Cancer.

This study aimed to evaluate AT1-R expression in normal and cancerous human kidneys, how these expressions are modified, and AT1-R functionality. AT-1R mRNA expression, determined by real-time PCR, was detected in all samples. AT-1R mRNA increased in well-differentiated cancer (G1, p < 0.01) and decreased 2.9-fold in undifferentiated cancer (G4, p < 0.001) compared with normal kidney tissues. Immunocytochemistry analysis showed that the AT-1R was expressed in the normal tubular epithelium. The glomerulus was also immunoreactive, and as expected, the smooth muscle cells of the vessel walls also expressed the receptor. A total of 35 out of 42 tumors were AT-1R positive, with the cell tumors showing varying numbers of immunoreactive cells, which were stained in a diffuse cytoplasmic and membranous pattern. Computer-assisted counting of the stained tumor cells showed that the number of AT-1R-positive cells increased in the well-differentiated cancers. The functionality of AT-1R was assessed in primary cultures of kidney epithelial cells obtained from three G3 kidney cancer tissues and corresponding histologically proven non-malignant tissue adjacent to the tumor. Indeed, Ang II stimulated, in a dose-dependent manner, the 24 h proliferation of normal kidney cells and cancer cells in the primary culture and phosphorylated extracellular regulated kinases 1 and 2. In conclusion, Ang II may be involved in the growth or function of neoplastic kidney tissue.

Clinical implication of tissue carcinoembryonic antigen expression in association with serum carcinoembryonic antigen in colorectal cancer

This study aimed to evaluate the prognostic significance of carcinoembryonic antigen (CEA) expression in tumor tissues of patients with colorectal cancer (CRC). The cohort included 7,412 patients with CRC from January 2010 to December 2015. Survival outcomes were assessed based on tissue CEA (t-CEA) patterns and intensities. Three-year (76.7% versus 81.3%) and 5-year (71.7% versus 77.6%, p < 0.001) disease-free survival (DFS) rates were significantly (p < 0.001) poorer in patients with a diffuse-cytoplasmic pattern than an apicoluminal pattern. Three-year (79% versus 86.6%) and 5-year (74.6% versus 84.7%) DFS rates were also significantly (p < 0.001) poorer in patients with high than low t-CEA intensity. Three-year (84.6% versus 88.4%) and 5-year (77.3% versus 82.6%) overall survival (OS) rates were significantly (p < 0.001) poorer in patients with diffuse-cytoplasmic than apicoluminal pattern of CEA expression, and both 3-year (86.7% versus 91.2%) and 5-year (80.1% versus 87.7%) OS rates were significantly (p < 0.001) poorer in patients with high than low t-CEA intensity. Multivariate analyses showed that high-intensity t-CEA was independently associated with DFS (p = 0.02; hazard ratio [HR] = 1.233) and OS (p = 0.032; HR = 1.228). Therefore, high-intensity t-CEA is a significant prognostic factor in CRC, independent of serum CEA (s-CEA), and can complement s-CEA in predicting survival outcomes after CRC resection.

Expression of the phagocytic receptors αMβ2 and αXβ2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells.

Activation of the integrin phagocytic receptors CR3 (αMβ2, CD11b/CD18) and CR4 (αXβ2, CD11c/CD18) requires Rap1 activation and RIAM function. RIAM controls integrin activation by recruiting Talin to β2 subunits, enabling the Talin-Vinculin interaction, which in term bridges integrins to the actin-cytoskeleton. RIAM also recruits VASP to phagocytic cups and facilitates VASP phosphorylation and function promoting particle internalization. Using a CRISPR-Cas9 knockout approach, we have analyzed the requirement for RIAM, VASP and Vinculin expression in neutrophilic-HL-60 cells. All knockout cells displayed abolished phagocytosis that was accompanied by a significant and specific reduction in ITGAM (αM), ITGAX (αX) and ITGB2 (β2) mRNA, as revealed by RT-qPCR. RIAM, VASP and Vinculin KOs presented reduced cellular F-actin content that correlated with αM expression, as treatment with the actin filament polymerizing and stabilizing drug jasplakinolide, partially restored αM expression. In general, the expression of αX was less responsive to jasplakinolide treatment than αM, indicating that regulatory mechanisms independent of F-actin content may be involved. The Serum Response Factor (SRF) was investigated as the potential transcription factor controlling αMβ2 expression, since its coactivator MRTF-A requires actin polymerization to induce transcription. Immunofluorescent MRTF-A localization in parental cells was primarily nuclear, while in knockouts it exhibited a diffuse cytoplasmic pattern. Localization of FHL-2 (SRF corepressor) was mainly sub-membranous in parental HL-60 cells, but in knockouts the localization was disperse in the cytoplasm and the nucleus, suggesting RIAM, VASP and Vinculin are required to maintain FHL-2 close to cytoplasmic membranes, reducing its nuclear localization and inhibiting its corepressor activity. Finally, reexpression of VASP in the VASP knockout resulted in a complete reversion of the phenotype, as knock-ins restored αM expression. Taken together, our results suggest that RIAM, VASP and Vinculin, are necessary for the correct expression of αMβ2 and αXβ2 during neutrophilic differentiation in the human promyelocytic HL-60 cell line, and strongly point to an involvement of these proteins in the acquisition of a phagocytic phenotype.

SmartFlareTM is a reliable method for assessing mRNA expression in single neural stem cells.

BACKGROUNDOne of the most challenging tasks of modern biology concerns the real-time tracking and quantification of mRNA expression in living cells. On this matter, a novel platform called SmartFlareTM has taken advantage of fluorophore-linked nanoconstructs for targeting RNA transcripts. Although fluorescence emission does not account for the spatial mRNA distribution, NanoFlare technology has grown a range of theranostic applications starting from detecting biomarkers related to diseases, such as cancer, neurodegenerative pathologies or embryonic developmental disorders.AIMTo investigate the potential of SmartFlareTM in determining time-dependent mRNA expression of prominin 1 (CD133) and octamer-binding transcription factor 4 (OCT4) in single living cells through differentiation.METHODSBrain fragments from the striatum of aborted human fetuses aged 8 wk postconception were processed to obtain neurospheres. For the in vitro differentiation, neurospheres were gently dissociated with Accutase solution. Single cells were resuspended in a basic medium enriched with fetal bovine serum, plated on poly-L-lysine-coated glass coverslips, and grown in a lapse of time from 1 to 4 wk. Live cell mRNA detection was performed using SmartFlareTM probes (CD133, Oct4, Actin, and Scramble). All the samples were incubated at 37 °C for 24 h. For nuclear staining, Hoechst 33342 was added. SmartFlareTM CD133- and OCT4-specific fluorescence signal was assessed using a semiquantitative visual approach, taking into account the fluorescence intensity and the number of labeled cells.RESULTSIn agreement with previous PCR experiments, a unique expression trend was observed for CD133 and OCT4 genes until 7 d in vitro (DIV). Fluorescence resulted in a mixture of diffuse cytoplasmic and spotted-like pattern, also detectable in the contacting neural branches. From 15 to 30 DIV, only few cells showed a scattered fluorescent pattern, in line with the differentiation progression and coherent with mRNA downregulation of these stemness-related genes.CONCLUSIONSmartFlareTM appears to be a reliable, easy-to-handle tool for investigating CD133 and OCT4 expression in a neural stem cell model, preserving cell biological properties in anticipation of downstream experiments.

Novel Imaging of Silver Nanoparticle Uptake by a Unicellular Alga and Trophic Transfer to Daphnia magna.

Widely applied silver nanoparticles (AgNPs) can have potentially detrimental impacts on aquatic organisms. Unicellular algae as primary producers can interact with AgNPs and initiate their transfer along food chains. Herein, we demonstrate that AgNPs were internalized in a freshwater phytoplankton species Chlamydomonas reinhardtii, but the entrance pathways varied with their surface coatings. Citrate-coated AgNPs (Cit-AgNPs) were internalized mainly through the apical zone of the cell near the flagella, whereas the aggregation-induced emission fluorogen (AIEgen)-coated AgNPs (AIE-AgNPs) were internalized through endocytosis. The internalized AgNPs were dissolved intracellularly and the released Ag+ was distributed heterogeneously in the cytoplasm, in contrast to the directly accumulated Ag+ which displayed a diffuse cytoplasmic distribution pattern. We then further visualized and quantified the trophic transfer of AgNPs from the alga C. reinhardtii to the zooplanktonic species Daphnia magna. Both trophically transferred Ag+ and AgNPs were concentrated in the gut regions of D. magna as a result of the direct ingestion of food particles. After ingestion, about 95% of the trophically transferred Ag+ was eliminated. Retention of AIE-AgNPs by daphnids was relatively higher than that of Cit-AgNPs due to their lower dissolution of Ag+. The present study provides direct evidence for the internalization of AgNPs in unicellular algae and demonstrates that the biological transport of trophically transferred of AgNPs is related to the different surface coatings of NPs.

MUC4 Expression in Angiomatoid Fibrous Histiocytoma.

Angiomatoid fibrous histiocytoma (AFH) is a rarely metastasizing neoplasm that typically occurs in the deep dermis and subcutis of the extremities of young patients, characterized by a t(2;22) translocation involving EWSR1 and CREB1. Because of its distinctive histologic features, the diagnosis of AFH is generally straightforward, although the immunohistochemistry (IHC) findings are relatively nonspecific. We recently encountered a case of primary cranial AFH that showed strong MUC4 IHC expression, which has not yet been reported previously. Prompted by this surprising finding, we investigated MUC4 expression in a series of AFH to evaluate this potential diagnostic pitfall. The expression of ALK by IHC, recently discovered in AFH, was also assessed in this study. We also analyzed EWSR1 rearrangement by fluorescence in situ hybridization using a dual color break-apart probe to confirm the diagnosis. The results showed MUC4 expression in 22.2% of AFH cases (4/18 cases), demonstrating a variable intensity of cytoplasmic staining. Most notably, one of the positive cases showed strong and diffuse expression. ALK IHC expression was observed in 17 of 18 cases (94.4%), usually in a diffuse and strong cytoplasmic pattern. EWSR1 rearrangement was demonstrated by fluorescence in situ hybridization in 81.2% of cases (13 of 16), including all the MUC4-positive cases. Our results indicate that although the significance of MUC4 expression in AFH is unknown, it is important to be aware that a subset of AFH can express the protein by IHC, expanding a variety of MUC4-positive mesenchymal tumors.

Loss of connexin43 in murine Sertoli cells and its effect on blood-testis barrier formation and dynamics.

Connexin43 (Cx43) is the predominant testicular gap junction protein and in cases of impaired spermatogenesis, Cx43 expression has been shown to be altered in several mammals. Amongst other functions, Cx43 is supposed to regulate junction formation of the blood-testis barrier (BTB). The aim of the present study was to investigate the expression pattern of different tight junction (TJ) proteins of the murine BTB using SC-specific Cx43 knockout mice (SCCx43KO). Adult homozygous male SCCx43KO mice (SCCx43KO-/-) predominantly show an arrest of spermatogenesis and SC-only tubules that might have been caused by an altered BTB assembly, composition or regulation. TJ molecules claudin-3, -5 and -11 were examined in adult wild type (WT) and SCCx43KO-/- mice using immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR). In this context, investigation of single tubules with residual spermatogenesis in SCCx43KO-/- mice was particularly interesting to identify a potential Cx43-independent influence of germ cells (GC) on BTB composition and dynamics. In tubules without residual spermatogenesis, a diffuse cytoplasmic distribution pattern for claudin-11 protein could be demonstrated in mutant mice. Nevertheless, claudin-11 seems to form functional TJ. Claudin-3 and -5 could not be detected immunohistochemically in the seminiferous epithelium of those tubules. Correspondingly, claudin-3 and -5 mRNA expression was decreased, providing evidence of generally impaired BTB dynamics in adult KO mice. Observations of tubules with residual spermatogenesis suggested a Cx43-independent regulation of TJ proteins by GC populations. To determine initial BTB formation in peripubertal SCCx43KO-/- mice, immunohistochemical staining and qRT-PCR of claudin-11 were carried out in adolescent SCCx43KO-/- and WT mice. Additionally, BTB integrity was functionally analysed using a hypertonic glucose fixative. These analyses revealed that SCCx43KO-/- mice formed an intact BTB during puberty in the same time period as WT mice, which however seemed to be accelerated.

Epithelioid cell histiocytoma with SQSTM1-ALK fusion: a case report

BackgroundEpithelioid cell histiocytoma (ECH), which is also known as epithelioid benign fibrous histiocytoma, has been classified as a rare variant of fibrous histiocytoma (FH). However, the recent detection of ALK protein expression and/or ALK gene rearrangement in ECH suggests that it might be biologically different from conventional FH.Case presentationA 27-year-old male presented with nodule on his left foot, which had been present for 5 years. A macroscopic examination revealed an exophytic, hyperkeratotic nodule on the dorsum of the left foot. Tumorectomy was performed, and a microscopic examination showed a subepidermal lesion composed of sheets of tumor cells with oval to round nuclei and ill-defined eosinophilic cytoplasm. The tumor cells were diffusely positive for factor XIIIa and ALK, but were negative for AE1/AE3 keratin, alpha-smooth muscle actin, CD30, CD34, CD68, PU.1, melan A, MITF, and S-100 protein. ALK immunostaining showed a diffuse cytoplasmic staining pattern. ALK fluorescence in situ hybridization demonstrated break-apart signals, which was suggestive of ALK rearrangement. A 5′-rapid amplification of cDNA ends assay detected SQSTM1-ALK fusion, in which exon 5 of the SQSTM1 gene was fused to exon 20 of the ALK gene. The patient was free from recurrence and distant metastasis at the 1-year of follow-up.ConclusionWe were able to demonstrate the SQSTM1-ALK fusion gene in ECH. Practically, detecting immunopositivity for ALK and appropriate cell-lineage markers are the key to diagnosing ECH.

Unusual Keratin Immunoprofile in a Rare Primary Pancreatic Ewing Sarcoma

Ewing Sarcoma/Peripheral Primitive Neuroectodermal Tumor is part of the small round blue cell tumors primarily occurring in bone and soft tissue, although rare cases in solid organs do exist. Diagnosis remains a challenge often requiring confirmation of a genetic translocation, as histopathology may be positive for a wide array of tumor markers sometimes causing misdiagnosis. We present an 11 year old female with primary pancreatic EWS/PNET, and an unusually broad cytokeratin profile. Our patient with no past medical or family history presented with abdominal pain, fatigue, and jaundice for 2 weeks. Physical exam noted jaundice, icterus, and a palpable non-tender RUQ mass. Labs showed normocytic anemia with elevated reticulocyte count, and LFTs indicative of an obstructive pattern. MRI confirmed a 7.4 x 6.2 x 4.8 cm mass centered within the pancreatic head/ampullary region, and invasion with tumor thrombus in the main portal vein. Endoscopy revealed a large antral impression from the tumor, with friable mucosa and active oozing in the duodenum. EUS displayed a pancreatic head mass abutting the common hepatic artery with invasion into the portal vein and a 22mm common bile duct. Biopsies were obtained from the friable duodenal tissue, with biliary stent placed for decompression. Duodenal biopsy showed a small round blue cell tumor with very scant cytoplasm, round hyperchromatic nuclei, finely dispersed chromatin and inconspicuous nucleoli embedded in a markedly desmoplastic fibrous stroma. Similar cells were seen from a pancreatic head mass FNA. Tumor cells were CD99 (MIC2) positive in a diffuse cytoplasmic pattern only. Immunohistochemistry had broad keratin positivity including cytokeratin CAM 5.2, cytokeratin AE1/AE3, cytokeratin 5/6, and cytokeratin 34BE12. P63, E-cadherin and CD117 (c-Kit) were also positive. FISH study and RT-PCR were positive for EWSR1-FLI1 fusion transcript and confirmed a EWS/ PNET, with a broad cytokeratin profile, primary to the pancreas. This is the first case of pancreatic PNET with immunohistochemistry showing such diffuse positivity in cytokeratin markers, and the 28th case of primary pancreatic PNET reported in the literature. CD99 is usually positive in membranous staining, however this case showed diffuse cytoplasmic positivity only, making the diagnosis challenging. This new pattern could reflect potential markers for prognostic studies or new targets for therapy.Figure: Keratin Cam 5.2 (20X): Positive in small round tumor cells.Figure. CD117: (20X): Positive in small round tumor cells.

The Networks of Genes Encoding Palmitoylated Proteins in Axonal and Synaptic Compartments Are Affected in PPT1 Overexpressing Neuronal-Like Cells.

CLN1 disease (OMIM #256730) is an early childhood ceroid-lipofuscinosis associated with mutated CLN1, whose product Palmitoyl-Protein Thioesterase 1 (PPT1) is a lysosomal enzyme involved in the removal of palmitate residues from S-acylated proteins. In neurons, PPT1 expression is also linked to synaptic compartments. The aim of this study was to unravel molecular signatures connected to CLN1. We utilized SH-SY5Y neuroblastoma cells overexpressing wild type CLN1 (SH-p.wtCLN1) and five selected CLN1 patients’ mutations. The cellular distribution of wtPPT1 was consistent with regular processing of endogenous protein, partially detected inside Lysosomal Associated Membrane Protein 2 (LAMP2) positive vesicles, while the mutants displayed more diffuse cytoplasmic pattern. Transcriptomic profiling revealed 802 differentially expressed genes (DEGs) in SH-p.wtCLN1 (as compared to empty-vector transfected cells), whereas the number of DEGs detected in the two mutants (p.L222P and p.M57Nfs*45) was significantly lower. Bioinformatic scrutiny linked DEGs with neurite formation and neuronal transmission. Specifically, neuritogenesis and proliferation of neuronal processes were predicted to be hampered in the wtCLN1 overexpressing cell line, and these findings were corroborated by morphological investigations. Palmitoylation survey identified 113 palmitoylated protein-encoding genes in SH-p.wtCLN1, including 25 ones simultaneously assigned to axonal growth and synaptic compartments. A remarkable decrease in the expression of palmitoylated proteins, functionally related to axonal elongation (GAP43, CRMP1 and NEFM) and of the synaptic marker SNAP25, specifically in SH-p.wtCLN1 cells was confirmed by immunoblotting. Subsequent, bioinformatic network survey of DEGs assigned to the synaptic annotations linked 81 DEGs, including 23 ones encoding for palmitoylated proteins. Results obtained in this experimental setting outlined two affected functional modules (connected to the axonal and synaptic compartments), which can be associated with an altered gene dosage of wtCLN1. Moreover, these modules were interrelated with the pathological effects associated with loss of PPT1 function, similarly as observed in the Ppt1 knockout mice and patients with CLN1 disease.

Combined use of nuclear phosphoprotein c-Myc and cellular phosphoprotein p53 for hepatocellular carcinoma detection in high-risk chronic hepatitis C patients

Background: Hepatocellular carcinoma (HCC) is a multistage process resulting from various genetic changes. We aimed to determine nuclear phosphoprotein c-Myc and cellular phosphoprotein p53 expression and to evaluate their importance in HCC diagnosis.Methods: One hundred and twenty chronic hepatitis C (CHC) patients (60 non-HCC CHC patients and 60 HCC patients who had a single small (<5 cm) tumour) were recruited. The gene products of c-Myc and p53 were identified in liver tissues and serum samples using immunostaining, western blot and ELISA.Results: Immunohistochemical detection of c-Myc and p53 with monospecific antibodies revealed intense and diffuse cytoplasmic staining patterns. Accumulated mutant proteins, released from tumour cells into the extracellular serum, were detected at 62 KDa, for c-Myc, and 53 KDa, for p53, using western blotting. In contrast to alpha feto-protein, there was a significant increase (p < 0.0001) in the positivity rate of c-Myc (86.7% vs. 6.7%) and p53 (78.3% vs. 8.3%) in the malignant vs. non-malignant patients. The parallel combination of c-Myc and p53 reach the absolute sensitivity (100%), for more accurate and reliable HCC detection (specificity was 87%).Conclusion: c-Myc and p53 are potential HCC diagnostic biomarkers, and convenient combinations of them could improve diagnostic accuracy of HCC.

Urinary proteins induce lysosomal membrane permeabilization and lysosomal dysfunction in renal tubular epithelial cells.

Lysosomal membrane permeabilization (LMP) has been shown to cause the release of cathepsins and other hydrolases from the lysosomal lumen to the cytosol and initiate a cell death pathway. Whether proteinuria triggers LMP in renal tubular epithelial cells (TECs) to accelerate the progression of renal tubulointerstitial injury remains unclear. In the present study, we evaluated TEC injury as well as changes in lysosomal number, volume, activity, and membrane integrity after urinary protein overload in vivo and in vitro. Our results revealed that neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 levels were significantly increased in the urine of patients with minimal change nephrotic syndrome (MCNS) and the culture supernatant of HK-2 cells treated by urinary proteins extracted from MCNS patients. Urinary protein overload also induced apoptotic cell death in HK-2 cells. Importantly, we found that lysosomal volume and number were markedly increased in TECs of patients with MCNS and HK-2 cells overloaded with urinary proteins. However, lysosome function, as assessed by proteolytic degradation of DQ-ovalbumin and cathepsin-B and cathepsin-L activities, was decreased in HK-2 cells overloaded with urinary proteins. Furthermore, urinary protein overload led to a diffuse cytoplasmic immunostaining pattern of cathepsin-B and irregular immunostaining of lysosome-associated membrane protein-1, accompanying a reduction in intracellular acidic components, which could be improved by pretreatment with antioxidant. Taken together, our results indicate that overloading of urinary proteins caused LMP and lysosomal dysfunction at least partly via oxidative stress in TECs.

Autophagy As a Target for Differentiation Therapy in Acute Myeloid Leukemia.

Abstract 2464Autophagy is a catabolic cellular process whereby redundant or damaged proteins and organelles are sequestered within vesicular “autophagosomes” and degraded to reusable monomers via lysosomal hydrolases. Autophagic activity is heightened in times of cellular stress or remodeling and has recently been proposed to play an important role in cell differentiation. As Acute Myeloid Leukemia (AML) is a condition that is characterized by a differentiation block, we have investigated the importance of autophagy in the differentiation of AML cells and the consequences of pharmacologic modification of autophagy on this process.We examined autophagy in the NB4 (t15:17, PML-RARα+) Acute Promyelocytic Leukemia (APML) cell line, a well-established model of leukemic cell differentiation. Treatment of NB4 cells for 4 days with all-trans-retinoic acid (ATRA) induced differentiation evidenced by cellular morphology and confirmed by both flow cytometric and western blot detection of the CD11b and CD14 surface markers of granulocytic differentiation.During days 0 to 4 of ATRA-induced differentiation, NB4 cells progressively accumulated cytoplasmic vesicles - a characteristic feature of autophagy. LC3, GABARAP and GATE16 are cytoplasmic proteins that become lipid-conjugated during autophagy activation and incorporated into autophagic vesicles. Immune-fluorescence microscopy demonstrated a shift in the staining pattern of these proteins from a diffuse cytoplasmic pattern to a discreet punctate pattern as differentiation proceeded, consistent with incorporation into autophagic vesicles. Up-regulation of these autophagy markers during differentiation was also confirmed by western blotting.To determine the functional consequences of autophagy inhibition, we treated NB4 cells with ATRA in the presence and absence of lysosomal inhibitors E64d and Pepstatin A. Expression of CD11b and CD14 was decreased at day 4 in cells treated with ATRA/E64/Pepstatin A compared to cells treated with ATRA alone. This was also seen by western blot.We next assessed the effects of Lithium, a known inducer of autophagy, on leukemic cell differentiation. We treated high passage HL60 cells (AML cell line more resistant to ATRA-induced differentiation, PML-RARα-) with Lithium (10mM) alone and in combination with ATRA (1μM) for 4 days. Western blot analysis confirmed that Lithium treatment elevated the expression of LC3, GABARAP and GATE16. We observed early morphological features of differentiation in combination-treated cells. CD11b expression at Day 4 was increased in Lithium-treated cells compared to cells treated with ATRA alone (24.1% v 12.4%). In combination-treated cells, differentiation was even further induced (33.5% CD11b expression). Combination treatment also resulted in increased apoptosis (Annexin V flow cytometric assay) at day 4 (22.7%) compared to Lithium alone (8.4%) and to ATRA alone (6.1%). Colony-forming assays were performed to assess the proliferative capacity of cells from these treatment groups sampled at day 2 and placed in drug-free methylcellulose medium for 7 days (MethoCult H4034). Colony numbers were significantly reduced in ATRA and Lithium combination-treated cells compared to either mono-therapy or controls.We have begun to test the use of Lithium alone and in combination with ATRA on primary AML samples. Early data suggests Lithium attenuates malignant cell growth on colony-forming assay. Experiments are underway to determine the molecular mechanisms whereby lithium sensitizes cells to ATRA and promotes differentiation. Conclusion:Autophagy plays a significant role in the myeloid differentiation of APL cells. Pharmacologic inhibition of autophagy impedes ATRA-induced leukemic cell differentiation while autophagy induction in non-APL leukemia in combination with ATRA promotes differentiation, decreases the proliferative capacity of malignant cells and decreases malignant cell viability. These findings suggest that autophagy induction with readily available agents such as Lithium may represent an exciting means of re-opening differentiation pathways in AML. We are continuing to test this hypothesis in AML patient samples. Disclosures:Orfali:MSD: Unrestricted educational support, Unrestricted educational support Other; Amgen: Unrestricted educational support Other; BMS: Unrestricted educational support, Unrestricted educational support Other; Novartis: Unrestricted educational support Other; Pfizer: Unrestricted educational support, Unrestricted educational support Other.

Equivocal p16 Immunostaining in Squamous Cell Carcinoma of the Head and Neck: Staining Patterns are Suggestive of HPV Status

p16 immunohistochemistry (IHC) is commonly used as a surrogate marker for human papillomavirus (HPV) detection in squamous cell carcinomas of the head and neck (SCCHN). However, the HPV status of tumors not staining strongly for p16 is difficult to interpret and may require the use of PCR, not available in all laboratories, as a final arbiter. We aim to determine if staining pattern in equivocal p16 staining and correlation to the percentage of positively stained tumor cells is predictive of HPV status. A retrospective review was performed on all SCCHN that underwent p16 IHC and PCR in our institution from 2007 to 2010. Descriptors of staining pattern in the original IHC report were retrieved. All available IHC slides were reviewed and reclassified using consensus staining pattern descriptors. Original and reclassified descriptors were compared to the final PCR HPV status for statistical significance using the χ(2) test. An estimate of the percentage of tumor cells that showed any form of staining was performed. Thirty-two SCCHN cases that underwent PCR HPV testing had equivocal p16 IHC results. Twenty-six cases available for review were reclassified into four staining patterns. Comparing age, sex, tumor site and diagnosis to HPV PCR status showed no statistically significant findings. However, comparing original descriptors to HPV status was statistically significant with isolated staining associated with negative HPV status (p=0.0002). Analysis using reclassified descriptors showed strong association of membranous/cytoplasmic staining of isolated cells with negative HPV status and faint, diffuse nuclear and cytoplasmic staining with positive HPV status (p=0.00006). HPV-negative cases with the former pattern had no more than 30% positively-stained tumor cells and HPV-positive cases with the latter pattern had 50-90% positively-stained cells. Our results suggest that pattern of staining in p16 IHC is associated with HPV status. For instance, a diffuse nuclear and cytoplasmic staining pattern, regardless of intensity, is associated with HPV positivity. The HPV-positive cases determined by staining pattern were also associated with a higher percentage of stained tumor cells.

Inhibin-A Immunoreactivity in Nervous System Lesions

To evaluate inhibin-A immunoreactivity and its utility in the differential diagnosis of nervous system neoplasms and non-neoplastic lesions. An immunohistochemical study of 252 central and peripheral nervous system tumors and 40 non-neoplastic lesions was undertaken. Brain lesions included the basic spectrum of astrocytic, oligodendroglial, and ependymal neoplasms, as well as glioneuronal, pineal parenchymal, choroid plexus, and embryonal. Meningeal neoplasms, basic peripheral nerve tumors, and uncommon sellar lesions were also assessed. Non-neoplastic lesions included demyelinating disease, progressive multifocal leukoencephalopathy, organizing infarct, and reactive gliosis. Diffuse cytoplasmic, membranous, and perinuclear cytoplasmic staining patterns were observed. Significant immunoreactivity was noted in glioblastoma (12 of 20), pleomorphic xanthoastrocytoma (6 of 10), ganglioglioma (8 of 10), meningioma (14 of 20), and hemangioblastoma (10 of 10). Peripheral nerve and sellar tumors as well as non-neoplastic lesions were entirely immunonegative. In our study that investigated the inhibin-A immunoreactivity in a broad spectrum of nervous system lesions, inhibin-A positivity was established in various low-grade and high-grade central nervous system tumors. Thus, inhibin-A is not a specific marker of hemangioblastoma and may be of limited utility in the differential diagnosis of astrocytic and meningothelial neoplasms. Its pathophysiologic role in these various tumors remains to be determined. Further evaluation of the possible significance of staining patterns and degrees of reactivity relative to pathobiology and/or prognosis significance is required.

Abstract 4105: Assessment of autophagosome formation in primary cells by lentiviral delivery of fluorescent protein-tagged LC3

Abstract Assessment of autophagosome formation has been greatly facilitated by monitoring redistribution of LC3-fluorescent protein fusions. The sensitivity of the technique and capability for real-time analysis of autophagosome formation are advantageous over detection of endogenous LC3 redistribution in many experimental settings. However, the necessity for transfection can be an impediment to analysis of autophagosome formation in difficult-to-transfect cells, particularly primary cultures. We have developed lentiviral vectors (employing 3rd generation technology for optimal biosafety) encoding monomeric TagGFP2 and TagRFP fused at the N-termini of wild-type LC3 and LC3G120A as tools for analysis of autophagy in a wide variety of cell types. In immortalized cell lines and in primary cells (HUVEC and human mesenchymal stem cells) transduced with the GFP- and RFP-LC3 lentiviruses, amino acid deprivation in the presence of a lysosome inhibitor induced redistribution of the fluorescence from a diffuse cytoplasmic pattern to a punctate distribution. Minimal background of punctae in fed cells was observed. 3-Methyladenine blocked the starvation-induced redistribution, and the G120A mutant did not undergo detectable redistribution upon amino acid deprivation. In addition, we demonstrate effectiveness of lentiviral GFP-LC3 in HUVECs by flow cytometry, in which the plasma membrane is selectively permeabilized such that free cytosolic fluorescent protein-tagged LC3 is released while autophagosome-bound LC3 fusion protein is retained. We anticipate that these lentiviral autophagy biosensors will facilitate analysis of autophagosome formation in physiologically relevant primary cells. 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 4105. doi:1538-7445.AM2012-4105

Cocaine and Certain of Its Long-Acting Metabolites Stimulate Endothelial Cell Von Willebrand Factor Secretion

Abstract 1148Cocaine ingestion is associated with increased risk of myocardial infarction, stroke, and deep vein thrombosis. Elevated risk for these thrombotic events persists for 7–10 days after cocaine ingestion, despite the fact that cocaine is rapidly cleared from the blood (with a half-life in circulation of less than 1 hour) being rapidly converted into numerous metabolites that circulate for 1–2 weeks. Thus, it is likely that these longer-lived cocaine metabolites account for the long-term thrombotic risk with cocaine use. Cocaine ingestion has been associated with increased plasma VWF concentration, early atherosclerosis, and platelet-rich arterial thrombi. VWF is the major adhesive ligand that attaches platelets to the vessel wall, either to the subendothelium at sites of injury where the endothelium has been denuded or, during inflammation, to intact endothelium, from which it is secreted. VWF is secreted by endothelial cells from intracellular storage granules (Weibel-Palade bodies) in a large, hyperadhesive multimeric form (ultra-large VWF, or ULVWF) that either remains tethered to the endothelial surface or is released into bulk flow. Platelet-VWF adhesion, and subsequent thrombus formation, may be augmented further by cocaine-induced vaso-constriction, increasing shear stress. Thus, we hypothesized that cocaine and/or its metabolites would stimulate endothelial VWF secretion as a mechanism of thrombotic risk. To test this possibility, we exposed cultured human endothelial cells from umbilical vein (HUVEC), brain microvasculature (BMVEC), or coronary artery (CAEC) to cocaine or one of its four major metabolites at concentration ranges reported to occur in plasma following cocaine use. The cocaine metabolites we tested were benzoylecgonine (BE), cocaethylene (CE), norcocaine (NC), and ecgonine methyl ester (EME). We assayed VWF release by platelet-VWF string formation in a parallel-plate flow chamber (2.5 dyne/cm2) and by measuring the concentration of VWF released into the supernatant. Cocaine concentrations as low as 0.1 μg/ml induced VWF release from HUVEC; 1–2 μg/ml cocaine was as effective in releasing VWF as 25 μM histamine or 4 mg/ml dDAVP. Of the cocaine metabolites, only BE and CE induced VWF release from endothelial cells. BMVEC were 10-fold more sensitive to cocaine and metabolites BE and CE than HUVEC in both platelet-string formation and VWF antigen assays. In CAEC, VWF release was slightly reduced compared to HUVEC in response to cocaine, BE, or CE. Consistent with this pattern, staining for intracellular VWF revealed the following hierarchy of intracellular VWF: BMVEC > HUVEC >> CAEC. In BMVEC and HUVEC, VWF staining was restricted to Weibel-Palade bodies, with CAEC also demonstrating a diffuse cytoplasmic pattern. We tested whether intracellular cAMP levels increased after cocaine or cocaine metabolite exposure to explore whether VWF release was dependent on Protein Kinase A, as is the case with dDAVP. Intracellular cAMP did not increase following exposure to cocaine or its metabolites in any of the endothelial cell lines. These results suggest that cocaine contributes to thrombosis by activating endothelial cells to secrete ULVWF via a mechanism that does not involve increased intracellular cAMP. The prolonged thrombotic risk after cocaine ingestion likely relates to the continued action of cocaine metabolites BE and/or CE on endothelial ULVWF secretion. VWF secretion is likely to vary between vascular beds, with brain endothelial cells being particularly sensitive. Furthermore, these results suggest that clinical management of cocaine-induced ischemia or vaso-occlusion may benefit from therapies aimed at disrupting the ULVWF–platelet interaction. Disclosures:No relevant conflicts of interest to declare.

MicroRNA expression patterns in lobular neoplasia.

10551 Background: Lobular carcinoma in situ (LCIS) is a marker of increased risk for subsequent development of invasive breast cancer. Molecular studies have identified a clonal relationship between LCIS and concurrent invasive lobular carcinoma (ILC), suggesting that LCIS may be a precursor lesion of ILC. We hypothesized that differences in miRNA expression patterns might exist in LCIS lesions with concurrent ILC compared with LCIS lesions found alone. Methods: RNA was collected from matched frozen and archival formalin fixed paraffin embedded (FFPE) breast tissue using TRIZOL extractions. MiRNA expression profiling was performed using the Illumina miRNA DASL assay, in duplicate experiments, allowing simultaneous profiling of 735 miRNAs. MiRNAs of interest were further validated and analyzed by quantitative RT-PCR (qrt-PCR) in triplicate. To verify our data by localizing specific miRNA sequences of interest, in situ hybridization was performed on FFPE sections using a one-day protocol with locked nucleic acid (LNA) detection probes for miR-375 and miR-182, the U6 small nuclear RNA probe as a positive control and a scrambled miRNA probe as a negative control. Results: qrt-PCR analysis of the miR-183/182/96 cluster and miR-375 was performed using normal breast tissue, LCIS alone, and matched synchronous LCIS and ILC. A trend of increasing miRNA expression correlating with a model of histological progression toward invasive disease was observed. Results of in-situ hybridization confirmed this trend, showing the weakest signal intensity for both miR-375 and miR-182 in normal breast tissue, a stronger signal in LCIS alone, and the strongest signal in LCIS associated with synchronous ILC. Localization of signal to the nucleus was observed in normal breast tissue and LCIS alone, while a more diffuse cytoplasmic localization pattern was observed in LCIS associated with ILC, possibly suggesting nucleocytoplasmic transport for further processing of these miRNAs during progression to a more invasive phenotype. Conclusions: Upregulation of miR-375 and the miR-183/182/96 cluster in LCIS may correlate with risk for progression to ILC. These novel biomarkers may also be potential targets for treatment.