ER-positive endocervical adenocarcinoma mimicking endometrioid adenocarcinoma in morphology and immunohistochemical profile: A case report of application of HPV RNAscope detection
Chen, R;Qin, P;Luo, Q;Yang, W;Tan, X;Cai, T;Jiang, Q;Chen, H;
PMID: 33787580 | DOI: 10.1097/MD.0000000000024927
Usual-type endocervical adenocarcinoma (ECA), high-risk HPV associated, is the most common type of glandular carcinoma in the endocervix. Mucin-depleted usual-type ECA is 1 end of morphological lineage of usual-type ECA and morphologically may show endometrioid features, which could cause diagnostic challenge with uterine endometrioid adenocarcinoma (EEC) and primary endometrioid ECA, especially in the setting of small biopsy and endocervical curettage (ECC). A 37-year-old women presented with dyspareunia for 1 year, showing atypical glandular cell on a liquid-based Pap TCT examination and positive for HPV16 detection. ECC showed EEC in another hospital based on its "endometrioid" morphology and immunohistochemical profiles (ER/PR/PAX8 strongly positive, though p16 also strongly positive). The specimen of hysterectomy in our hospital displayed a lesion confined to the uterine cervix showing the same morphology and immunohistochemical profiles as ECC. Finally, we successfully performed HPV RNAscope and detected high-risk human papilloma virus (HPV) E6/E7 mRNA particles in tumor cells in situ, which warranted usual-type ECA with mucin-depleted feature, a rare deviation of usual-type of ECA. The patient underwent total hysterectomy with lymph node dissection. To date, 14 months after surgery, the patient is well without recurrence or distant metastasis, and undergoes regular reexamination. We report a rare case of mucin-depleted usual-type ECA showing overlapping morphological and immunohistochemical profiles with EEC. The pathological diagnosis was confirmed by high-risk HPV RNAscope detection which is superior than immunohistochemistry to identify usual-type ECA, warranting an important role in assisting the diagnosis of morphological vague cases.
Prostate, 73(9):1007–1015.
Shinohara DB, Vaghasia AM, Yu SH, Mak TN, Brüggemann H, Nelson WG, De Marzo AM, Yegnasubramanian S, Sfanos KS (2013).
PMID: 23389852 | DOI: 10.1002/pros.22648.
BACKGROUND:
Prostatic inflammation has been linked to a number of prostatic diseases such as benign prostatic hyperplasia (BPH), prostatitis syndromes, and prostate cancer. Major unanswered questions include what pathogenic mechanisms, such as bacterial infections, may drive the accumulation of inflammatory infiltrates in the human prostate, and how inflammation might contribute to disease. To study this potential link in an in vivo system, we developed a mouse model of long-term bacteria-induced chronic inflammation of the prostate using a human prostatectomy-derived strain of Propionibacterium acnes.
METHODS:
C57BL/6J mice were inoculated, via urethral catheterization, with vehicle control or a prostatectomy-derived strain of P. acnes (PA2). Animals were assessed at 2 days, 1, 2, or 8 weeks post-inoculation via histology and immunohistochemistry (IHC).
RESULTS:
PA2 inoculation resulted in severe acute and chronic inflammation confined to the dorsal lobe of the prostate. Chronic inflammation persisted for at least 8 weeks post-inoculation. Inflammatory lesions were associated with an increase in the Ki-67 proliferative index, and diminished Nkx3.1 and androgen receptor (AR) production. Interestingly, the observed response required live bacteria and both IHC and in situ hybridization assays for P. acnes indicated a potential intracellular presence of P. acnes in prostate epithelial cells.
CONCLUSIONS:
To our knowledge, this is the first mouse model of long-term prostatic inflammation induced by P. acnes, and more generally, any prostatectomy-derived bacterial isolate. This model may serve as a valuable preclinical model of chronic prostatic inflammation that can be used to mechanistically study the link between inflammation and prostatic disease.
Pillai SG, Zhu P, Siddappa CM, Adams DL, Li S, Makarova OV, Amstutz P, Nunley R, Tang CM, Watson MA, Aft RL.
PMID: 28129357 | DOI: 10.1371/journal.pone.0170761
Abstract
PURPOSE:
Molecular characterization of disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer (BC) patients has been hindered by their rarity. To enrich for these cells using an antigen-independent methodology, we have evaluated a size-based microfiltration device in combination with several downstream biomarker assays.
METHODS:
BM aspirates were collected from healthy volunteers or BC patients. Healthy BM was mixed with a specified number of BC cells to calculate recovery and fold enrichment by microfiltration. Specimens were pre-filtered using a 70 μm mesh sieve and the effluent filtered through CellSieve microfilters. Captured cells were analyzed by immunocytochemistry (ICC), FISH for HER-2/neu gene amplification status, and RNA in situ hybridization (RISH). Cells eluted from the filter were used for RNA isolation and subsequent qRT-PCR analysis for DTC biomarker gene expression.
RESULTS:
Filtering an average of 14×106 nucleated BM cells yielded approximately 17-21×103 residual BM cells. In the BC cell spiking experiments, an average of 87% (range 84-92%) of tumor cells were recovered with approximately 170- to 400-fold enrichment. Captured BC cells from patients co-stained for cytokeratin and EpCAM, but not CD45 by ICC. RNA yields from 4 ml of patient BM after filtration averaged 135ng per 10 million BM cells filtered with an average RNA Integrity Number (RIN) of 5.3. DTC-associated gene expression was detected by both qRT-PCR and RISH in filtered spiked or BC patient specimens but, not in control filtered normal BM.
CONCLUSIONS:
We have tested a microfiltration technique for enrichment of BM DTCs. DTC capture efficiency was shown to range from 84.3% to 92.1% with up to 400-fold enrichment using model BC cell lines. In patients, recovered DTCs can be identified and distinguished from normal BM cells using multiple antibody-, DNA-, and RNA-based biomarker assays.
Nakajima N, Yoshizawa A, Kondo K, Rokutan-Kurata M, Hirata M, Furuhata A, Sumiyoshi S, Sonobe M, Menju T, Momose M, Fujimoto M, Date H, Haga H.
PMID: 28231386 | DOI: 10.1111/his.13198
Abstract
AIMS:
An easy and rapid assay for detecting mRNA in formalin-fixed paraffin-embedded samples (RNA-in situ hybridization; RNA-ISH) has been reported recently. We investigated the diagnostic accuracy of RNA-ISH for detecting lung adenocarcinoma (LA) with anaplastic lymphoma kinase (ALK) gene rearrangement.
METHODS AND RESULTS:
We tested ALK RNA-ISH on 11 resected LAs for which ALK fusion was confirmed by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH). ALK mRNA expression was detected by RNA-ISH in all 11 ALK-positive LAs, with a mean positive cell proportion of 68.4% (median, 75.3%; range, 3-98.8%) by counting 100 tumor cells at 10 different loci; RNA-ISH did not detect ALK mRNA expression in the normal surrounding lung cells. Next, we explored the concordance between ALK RNA-ISH and IHC/FISH tests using tissue microarrays (TMAs) containing 294 LAs. In the TMA slides, we found 5 ALK-positive cases via IHC and/or FISH. The mean proportion of ALK RNA-ISH-positive cells in these 5 cases was 75.6% (median, 82%; range, 40-94%), whereas the proportion of ALK RNA-ISH-positive cells in the remaining 289 cases was 0.3% (median 0; range, 0-15%). When the cutoff value was set at 15%, ALK RNA-ISH-positive and ALK RNA-ISH-negative cases were distinguishable with 100% sensitivity and specificity relative to the IHC/FISH tests.
CONCLUSIONS:
Our findings show that RNA-ISH is useful for detecting ALK rearrangement with high sensitivity and specificity relative to conventional IHC/FISH tests. Thus, RNA-ISH, which is an easy and rapid assay, can be an alternative method to IHC and FISH. This article is protected by copyright. All rights reserved.
Am J Physiol Renal Physiol.
Herrera M, Söderberg M, Sabirsh A, Valastro B, Mölne J, Santamaria B, Valverde AM, Guionaud S, Heasman S, Bigley A, Jermutus L, Rondinone C, Coghlan M, Baker D, Quinn CM.
PMID: 27440778 | DOI: 10.1152/ajprenal.00179.2016
Diabetic nephropathy (DN) remains an unmet medical challenge as its prevalence is projected to continue to increase and specific medicines for treatment remain undeveloped. Activation of the immune system, in particular T-cells, is emerging as a possible mechanism underlying DN disease progression in humans and animal models. We hypothesized that inhibition of T-cell activation will ameliorate DN. Interaction of B7-1 (CD80) on the surface of antigen presenting cells with its binding partners, CTLA4 (CD152) and CD28 on T-cells, is essential for T-cell activation. In this study we used the soluble CTLA4-Fc fusion protein Abatacept to block cell surface B7-1, preventing the cellular interaction and inhibiting T-cell activation. When Abatacept was dosed in an animal model of diabetes-induced albuminuria, it reduced albuminuria in both prevention and intervention modes. The number of T-cells infiltrating the kidneys of DN animals correlated with the degree of albuminuria, and treatment with Abatacept reduced the number of renal T-cells. As B7-1 induction has been recently proposed to underlie podocyte damage in DN, Abatacept could be efficacious in DN by protecting podocytes. However, this does not appear to be the case as B7-1 was not expressed in 1) kidneys of DN animals; 2) stimulated human podocytes in culture; or 3) glomeruli of DN patients. We conclude that Abatacept ameliorates DN by blocking systemic T-cell activation and not by interacting with podocytes.
Rozo AV, Babu DA, Suen PA, Groff DN, Seeley RJ, Simmons RA, Seale P, Ahima RS, Stoffers DA.
PMID: - | DOI: 10.1016/j.molmet.2017.05.006
Objective
Adult obesity risk is influenced by alterations to fetal and neonatal environments. Modifying neonatal gut or neurohormone signaling pathways can have negative metabolic consequences in adulthood. Here we characterize the effect of neonatal activation of glucagon like peptide-1 (GLP-1) receptor (GLP1R) signaling on adult adiposity and metabolism.
Methods
Wild type C57BL/6 mice were injected with 1 nmol/kg Exendin-4 (Ex-4), a GLP1R agonist, for 6 consecutive days after birth. Growth, body composition, serum analysis, energy expenditure, food intake, and brain and fat pad histology and gene expression were assessed at multiple time points through 42 weeks. Similar analyses were conducted in a Glp1r conditional allele crossed with a Sim1Cre deleter strain to produce Sim1Cre;Glp1rloxP/loxP mice and control littermates.
Results
Neonatal administration of Ex-4 reduced adult body weight and fat mass, increased energy expenditure, and conferred protection from diet-induced obesity in female mice. This was associated with induction of brown adipose genes and increased noradrenergic fiber density in parametrial white adipose tissue (WAT). We further observed durable alterations in orexigenic and anorexigenic projections to the paraventricular hypothalamic nucleus (PVH). Genetic deletion of Glp1r in the PVH by Sim1-Cre abrogated the impact of neonatal Ex-4 on adult body weight, WAT browning, and hypothalamic architecture.
Conclusion
These observations suggest that the acute activation of GLP1R in neonates durably alters hypothalamic architecture to limit adult weight gain and adiposity, identifying GLP1R as a therapeutic target for obesity prevention.
J Biol Chem. 2018 Oct 12.
El Jellas K, Johansson BB, Fjeld K, Antonopoulos A, Immervoll H, Choi MH, Hoem D, Lowe ME, Lombardo D, Njølstad PR, Dell A, Mas E, Haslam SM, Molven A.
PMID: 30315106 | DOI: 10.1074/jbc.RA118.001934
Carboxyl-ester lipase (CEL) is a pancreatic fat-digesting enzyme associated with human disease. Rare mutations in the CEL gene cause a syndrome of pancreatic exocrine and endocrine dysfunction denoted MODY8, whereas a recombined CEL allele increases the risk for chronic pancreatitis. Moreover, CEL has been linked to pancreatic ductal adenocarcinoma (PDAC) through a postulated oncofetal CEL variant termed feto-acinar pancreatic protein (FAPP). The monoclonal antibody mAb16D10 was previously reported to detect a glycotope in the highly O-glycosylated, mucin-like C-terminus of CEL/FAPP. We here assessed the expression of human CEL in malignant pancreatic lesions and cell lines. CEL was not detectably expressed in neoplastic cells, implying that FAPP is unlikely to be a glycoisoform of CEL in pancreatic cancer. Testing of the mAb16D10 antibody in glycan microarrays then demonstrated that it recognized structures containing terminal GalNAc-α1,3(Fuc-α1,2)Gal (blood group A antigen) and also repeated protein sequences containing GalNAc residues linked to Ser/Thr (Tn antigen), findings that were supported by immunostainings of human pancreatic tissue. To examine whether the CEL glycoprotein might be modified by blood group antigens, we used high-sensitivity MALDI-TOF mass spectrometry to characterize the released O-glycan pool of CEL immunoprecipitated from human pancreatic juice. We found that the O-glycome of CEL consisted mainly of core 1/2 structures with a composition depending on FUT2 and ABO gene polymorphisms. Thus, among digestive enzymes secreted by the pancreas, CEL is a glycoprotein with some unique characteristics, supporting the view that it could serve additional biological functions to its cholesteryl esterase activity in the duodenum.
He, S;Lei, P;Kang, W;Cheung, P;Xu, T;Mana, M;Park, C;Wang, H;Imada, S;Russell, J;Wang, J;Wang, R;Zhou, Z;Chetal, K;Stas, E;Mohad, V;Bruun-Rasmussen, P;Sadreyev, R;Hodin, R;Zhang, Y;Breault, D;Camargo, F;Yilmaz, Ö;Fredberg, J;Saeidi, N;
| DOI: 10.1053/j.gastro.2023.02.030
Background & aims Fibrosis and tissue stiffening are hallmarks of the inflammatory bowel disease (IBD). We have hypothesized that the increased stiffness directly contributes to the dysregulation of the epithelial cell homeostasis in IBD. Here, we aim to determine the impact of tissue stiffening on the fate and function of the intestinal stem cells (ISCs). Methods We developed a long-term culture system consisting of 2.5-dimensional intestinal organoids grown on a hydrogel matrix with tunable stiffness. Single-cell RNA sequencing provided stiffness-regulated transcriptional signatures of the ISCs and their differentiated progeny. YAP-knockout and YAP-overexpression mice were used to manipulate YAP expression. In addition, we analyzed colon samples from murine colitis models and human IBD samples to assess the impact of stiffness on ISCs in vivo. Results We demonstrated that increasing the stiffness potently reduced the population of LGR5+ ISCs and KI-67+ proliferating cells. Conversely, cells expressing the stem cell marker, OLFM4, became dominant in the crypt-like compartments and pervaded the villus-like regions. Concomitantly, stiffening prompted the ISCs to preferentially differentiate toward goblet cells. Mechanistically, stiffening increased the expression of cytosolic YAP, driving the extension of OLFM4+ cells into the villus-like regions, while it induced the nuclear translocation of YAP, leading to preferential differentiation of ISCs towards goblet cells. Furthermore, analysis of colon samples from murine colitis models and IBD patients demonstrated cellular and molecular remodeling reminiscent of those observed in vitro. Conclusions Collectively, our findings highlight that matrix stiffness potently regulates the stemness of ISCs and their differentiation trajectory, supporting the hypothesis that fibrosis-induced gut stiffening plays a direct role in epithelial remodeling in IBD.
Am J Surg Pathol. Dec;36(12):1874–1882.
Bishop JA, Ma XJ, Wang H, Luo Y, Illei PB, Begum S, Taube JM, Koch WM, Westra WH (2012).
PMID: 23060353 | DOI: 10.1097/PAS.0b013e318265fb2b.
Evidence for transcriptional activation of the viral oncoproteins E6 and E7 is regarded as the gold standard for the presence of clinically relevant human papillomavirus (HPV), but detection of E6/E7 mRNA requires RNA extraction and polymerase chain reaction amplification-a challenging technique that is restricted to the research laboratory. The development of RNA in situ hybridization (ISH) probes complementary to E6/E7 mRNA permits direct visualization of viral transcripts in routinely processed tissues and has opened the door for accurate HPV detection in the clinical care setting. Tissue microarrays containing 282 head and neck squamous cell carcinomas from various anatomic subsites were tested for the presence of HPV using p16 immunohistochemistry, HPV DNA ISH, and an RNA ISH assay (RNAscope) targeting high-risk HPV E6/E7 mRNA transcripts. The E6/E7 mRNA assay was also used to test an additional 25 oropharyngeal carcinomas in which the HPV status as recorded in the surgical pathology reports was equivocal due to conflicting detection results (ie, p16 positive, DNA ISH negative). By the E6/E7 mRNA method, HPV was detected in 49 of 282 (17%) HNSCCs including 43 of 77 (56%) carcinomas from the oropharynx, 2 of 3 (67%) metastatic HNSCCs of an unknown primary site, 2 of 7 (29%) carcinomas from the sinonasal tract, and 2 of 195 (1%) carcinomas from other head and neck sites. p16 expression was strongly associated with the presence of HPV E6/E7 mRNA: 46 of 49 HPV-positive tumors exhibited p16 expression, whereas only 22 of 233 HPV-negative tumors were p16 positive (94% vs. 9%, P<0.0001). There was also a high rate of concordance (99%) between the E6/E7 mRNA method and HPV DNA ISH. For the selected group of discordant HNSCCs (p16/HPV DNA), the presence of E6/E7 transcripts was detected in 21 of 25 (84%) cases. The E6/E7 mRNA method confirmed the presence of transcriptionally active HPV-related HNSCC that has a strong predilection for the oropharynx and is strongly associated with high levels of p16 expression. Testing for HPV E6/E7 transcripts by RNA ISH is ideal because it confirms the presence of integrated and transcriptionally active virus, permits visualization of viral transcripts in tissues, and is technically feasible for routine testing in the clinical laboratory.
Breast cancer research : BCR
Mir, S;Golden, BDO;Griess, BJ;Vengoji, R;Tom, E;Kosmacek, EA;Oberley-Deegan, RE;Talmon, GA;Band, V;Teoh-Fitzgerald, ML;
PMID: 35836253 | DOI: 10.1186/s13058-022-01548-6
A pro-oxidant enzyme, NADPH oxidase 4 (Nox4) has been reported to be a critical downstream effector of TGFβ-induced myofibroblast transformation during fibrosis. While there are a small number of studies suggesting an oncogenic role of Nox4 derived from activated fibroblasts, direct evidence linking this pro-oxidant to the tumor-supporting CAF phenotype and the mechanisms involved are lacking, particularly in breast cancer.We targeted Nox4 in breast patient-derived CAFs via siRNA-mediated knockdown or administration of a pharmaceutical inhibitor (GKT137831). We also determine primary tumor growth and metastasis of implanted tumor cells using a stable Nox4-/- syngeneic mouse model. Autophagic flux of CAFs was assessed using a tandem fluorescent-tagged ptfl-LC3 plasmid via confocal microscopy analysis and determination of the expression level of autophagy markers (beclin-1 and LC3B). Nox4 overexpressing CAFs depend on the Nrf2 (nuclear factor-erythroid factor 2-related factor 2) pathway for survival. We then determined the dependency of Nox4-overexpressing CAFs on the Nrf2-mediated adaptive stress response pathway for survival. Furthermore, we investigated the involvement of Birc5 on CAF phenotype (viability and collagen contraction activity) as well as the expression level of CAF markers, FAP and αSMA.We found that deletion of stroma Nox4 and pharmaceutically targeting its activity with GKT137831 significantly inhibited orthotopic tumor growth and metastasis of implanted E0771 and 4T1 murine mammary carcinoma cell lines in mice. More importantly, we found a significant upregulation of Nox4 expression in CAFs isolated from human breast tumors versus normal mammary fibroblasts (RMFs). Our in situ RNA hybridization analysis for Nox4 transcription on a human breast tumor microarray further support a role of this pro-oxidant in the stroma of breast carcinomas. In addition, we found that Nox4 promotes autophagy in CAFs. Moreover, we found that Nox4 promoted survival of CAFs via activation of Nrf2, a master regulator of oxidative stress response. We have further shown Birc5 is involved as a downstream modulator of Nrf2-mediated pro-survival phenotype. Together these studies indicate a role of redox signaling via the Nox4-Nrf2 pathway in tumorigenesis and metastasis of breast cancer cells by promoting autophagy and survival of CAFs.
Molecular Vision 2015; 21:428-442
Sun N, Shibata B, Hess JF, FitzGerald PG.
PMID: 25991907
Purpose: Several properties of ocular tissue make fixation for light microscopy problematic. Because the eye is spherical, immersion fixation necessarily results in a temporal gradient of fixation, with surfaces fixing more rapidly and thoroughly than interior structures. The problem is compounded by the fact that the layers of the eye wall are compositionally quite different, resulting in different degrees of fixation-induced shrinkage and distortion. Collectively, these result in non-uniform preservation, as well as buckling and/or retinal detachment. This gradient problem is most acute for the lens, where the density of proteins can delay fixation of the central lens for days, and where the fixation gradient parallels the age gradient of lens cells, which complicates data interpretation. Our goal was to identify a simple method for minimizing some of the problems arising from immersion fixation, which avoided covalent modification of antigens, retained high quality structure, and maintained tissue in a state that is amenable to common cytochemical techniques. Methods: A simple and inexpensive derivative of the freeze-substitution approach was developed and compared to fixation by immersion in formalin. Preservation of structure, immunoreactivity, GFP and tdTomato fluorescence, lectin reactivity, outer segment auto fluorescence, Click-iT chemistry, compatibility with in situ hybdrdization, and the ability to rehydrate eyes after fixation by freeze substitution for subsequent cryo sectioning were assessed. Results: An inexpensive and simple variant of the freeze substitution approach provides excellent structural preservation for light microscopy, and essentially eliminates ocular buckling, retinal detachment, and outer segment auto-fluorescence, without covalent modification of tissue antigens. The approach shows a notable improvement in preservation of immunoreactivity. TdTomato intrinsic fluorescence is also preserved, as is compatibility with in situ hybridization, lectin labeling, and the Click-iT chemistry approach to labeling the thymidine analog EdU. On the negative side, this approach dramatically reduced intrinsic GFP fluorescence. Conclusions: A simple, cost-effective derivative of the freeze substitution process is described that is of particular value in the study of rodent or other small eyes, where fixation gradients, globe buckling, retinal detachment, differential shrinkage, autofluorescence, and tissue immunoreactivity have been problematic.
Qu X, Sandmann T, Frierson H Jr, Fu L, Fuentes E, Walter K, Okrah K, Rumpel C, Moskaluk C, Lu S, Wang Y, Bourgon R, Penuel E, Pirzkall A, Amler L, Lackner MR, Tabenero J, Hampton GM, Kabbarah O.
PMID: 27270421 | DOI: 10.1038/onc.2016.170.
Key molecular drivers that underlie transformation of colonic epithelium into colorectal adenocarcinoma (CRC) are well described. However, the mechanisms through which clinically targeted pathways are activated during CRC progression have yet to be elucidated. Here, we used an integrative genomics approach to examine CRC progression. We used laser capture microdissection to isolate colonic crypt cells, differentiated surface epithelium, adenomas, carcinomas and metastases, and used gene expression profiling to identify pathways that were differentially expressed between the different cell types. We identified a number of potentially important transcriptional changes in developmental and oncogenic pathways, and noted a marked upregulation of EREG in primary and metastatic cancer cells. We confirmed this pattern of gene expression by in situ hybridization and observed staining consistent with autocrine expression in the tumor cells. Upregulation of EREG during the adenoma-carcinoma transition was associated with demethylation of two key sites within its promoter, and this was accompanied by an increase in the levels of epidermal growth factor receptor (EGFR) phosphorylation, as assessed by reverse-phase protein analysis. In CRC cell lines, we demonstrated that EREG demethylation led to its transcriptional upregulation, higher levels of EGFR phosphorylation, and sensitization to EGFR inhibitors. Low levels of EREG methylation in patients who received cetuximab as part of a phase II study were associated with high expression of the ligand and a favorable response to therapy. Conversely, high levels of promoter methylation and low levels of EREG expression were observed in tumors that progressed after treatment. We also noted an inverse correlation between EREG methylation and expression levels in several other cancers, including those of the head and neck, lung and bladder. Therefore, we propose that upregulation of EREG expression through promoter demethylation might be an important means of activating the EGFR pathway during the genesis of CRC and potentially other cancers.
