A six-year comprehensive review of eye cytology cases received at a tertiary level hospital.

BACKGROUND: Ocular cytology is an effective method of diagnosing infective, benign, and malignant ocular disease processes due to easy accessibility and rapid turnaround time. However, these specimens pose significant diagnostic challenges due to rarity of the specimen type, sparse diagnostic material available for ancillary workup, and unfamiliarity of the diagnostic entities by the cytopathologist. METHODS: This study conducted a 6-year comprehensive review of 65 eye cytology cases received at a tertiary level hospital. Cytopathologic diagnoses of "negative for malignancy" and "atypical" were categorized as negative findings (70.8%, n = 46) and diagnoses of "suspicious for malignancy" and "positive for malignancy" were categorized as positive findings (23.1%, n = 15). A 44.6% (n = 29) of cases had subsequent histopathology and/or flow cytometry diagnoses. Premalignant and malignant lesions detected on histopathology were considered as significant findings. Statistical analysis was performed to evaluate the concordance of ocular cytology with associated histopathology and/or flow cytometry diagnoses. RESULTS: The accuracy of final cytology-histopathology and/or cytology-flow cytometry diagnoses in this cohort of cases is 86.2%. The sensitivity and specificity of ocular diagnosis by cytology are 66.6% and 100%, respectively. The positive and negative predictive values of ocular diagnosis by cytology are 100% and 80.9%, respectively. CONCLUSION: Ocular cytology is a fast, effective, and sensitive method for diagnosing ocular pathology specimens. Familiarity with these specimen types by cytopathologists can help in diagnosing ocular diseases effectively on small, challenging cytologic preparations.

Erythrophagocytosis is not a reproducible finding in liver biopsies, and is not associated with clinical diagnosis of hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a rare disease with high mortality. Liver involvement is common (based on elevated liver function tests) with most patients demonstrating acute hepatitis. Liver biopsies are frequently obtained in the setting of suspected HLH for the purpose of identification of erythrophagocytosis, and if present, this finding is thought to suggest or support the diagnosis of HLH. However, there are problems with this approach; in particular, we do not know whether this finding is reproducible or whether it is specific to HLH. Therefore, we conducted a multi-institutional study in which experienced liver pathologists reviewed images taken from liver biopsies from patients with normal liver, acute hepatitis, possible HLH, and clinical HLH to determine if there was agreement about the presence or absence of erythrophagocytosis, and to ascertain whether the finding corresponds to a clinical diagnosis of HLH. Twelve liver pathologists reviewed 141 images in isolation (i.e., no clinical information or diagnosis provided). These came from 32 patients (five normal, 17 acute hepatitis, six HLH, four possible HLH). The pathologists classified each image as negative, equivocal, or positive for erythrophagocytosis. Kappa was .08 (no agreement) for case-level and 0.1 for image-level (1.4% agreement, based on two images which were universally considered negative). There was no difference in the proportion of pathologists who diagnosed erythrophagocytosis among those with different diagnoses at case or image-level (p = 0.82 and p = 0.82, respectively). Thus, erythrophagocytosis is an entirely unreliable histologic parameter in liver, as it is irreproducible and not demonstrably associated with a clinical disease (namely, HLH). Unless and until more reliable guidelines can be established, pathologists should refrain from commenting on the presence or absence of erythrophagocytosis in liver biopsy.

Targeting breast and pancreatic cancer metastasis using a dual-cadherin antibody.

The successful application of antibody-based therapeutics in either primary or metastatic cancer depends upon the selection of rare cell surface epitopes that distinguish cancer cells from surrounding normal epithelial cells. By contrast, as circulating tumor cells (CTCs) transit through the bloodstream, they are surrounded by hematopoietic cells with dramatically distinct cell surface proteins, greatly expanding the number of targetable epitopes. Here, we show that an antibody (23C6) against cadherin proteins effectively suppresses blood-borne metastasis in mouse isogenic and xenograft models of triple negative breast and pancreatic cancers. The 23C6 antibody is remarkable in that it recognizes both the epithelial E-cadherin (CDH1) and mesenchymal OB-cadherin (CDH11), thus overcoming considerable heterogeneity across tumor cells. Despite its efficacy against single cells in circulation, the antibody does not suppress primary tumor formation, nor does it elicit detectable toxicity in normal epithelial organs, where cadherins may be engaged within intercellular junctions and hence inaccessible for antibody binding. Antibody-mediated suppression of metastasis is comparable in matched immunocompetent and immunodeficient mouse models. Together, these studies raise the possibility of antibody targeting CTCs within the vasculature, thereby suppressing blood-borne metastasis.

Satellite repeat RNA expression in epithelial ovarian cancer associates with a tumor-immunosuppressive phenotype.

Aberrant expression of viral-like repeat elements is a common feature of epithelial cancers, and the substantial diversity of repeat species provides a distinct view of the cancer transcriptome. Repeatome profiling across ovarian, pancreatic, and colorectal cell lines identifies distinct clustering independent of tissue origin that is seen with coding gene analysis. Deeper analysis of ovarian cancer cell lines demonstrated that human satellite II (HSATII) satellite repeat expression was highly associated with epithelial-mesenchymal transition (EMT) and anticorrelated with IFN-response genes indicative of a more aggressive phenotype. SATII expression - and its correlation with EMT and anticorrelation with IFN-response genes - was also found in ovarian cancer RNA-Seq data and was associated with significantly shorter survival in a second independent cohort of patients with ovarian cancer. Repeat RNAs were enriched in tumor-derived extracellular vesicles capable of stimulating monocyte-derived macrophages, demonstrating a mechanism that alters the tumor microenvironment with these viral-like sequences. Targeting of HSATII with antisense locked nucleic acids stimulated IFN response and induced MHC I expression in ovarian cancer cell lines, highlighting a potential strategy of modulating the repeatome to reestablish antitumor cell immune surveillance.

Reverse Transcriptase Inhibition Disrupts Repeat Element Life Cycle in Colorectal Cancer.

Altered RNA expression of repetitive sequences and retrotransposition are frequently seen in colorectal cancer, implicating a functional importance of repeat activity in cancer progression. We show the nucleoside reverse transcriptase inhibitor 3TC targets activities of these repeat elements in colorectal cancer preclinical models with a preferential effect in p53-mutant cell lines linked with direct binding of p53 to repeat elements. We translate these findings to a human phase II trial of single-agent 3TC treatment in metastatic colorectal cancer with demonstration of clinical benefit in 9 of 32 patients. Analysis of 3TC effects on colorectal cancer tumorspheres demonstrates accumulation of immunogenic RNA:DNA hybrids linked with induction of interferon response genes and DNA damage response. Epigenetic and DNA-damaging agents induce repeat RNAs and have enhanced cytotoxicity with 3TC. These findings identify a vulnerability in colorectal cancer by targeting the viral mimicry of repeat elements. SIGNIFICANCE: Colorectal cancers express abundant repeat elements that have a viral-like life cycle that can be therapeutically targeted with nucleoside reverse transcriptase inhibitors (NRTI) commonly used for viral diseases. NRTIs induce DNA damage and interferon response that provide a new anticancer therapeutic strategy. This article is highlighted in the In This Issue feature, p. 1397.

A non-dividing cell population with high pyruvate dehydrogenase kinase activity regulates metabolic heterogeneity and tumorigenesis in the intestine.

Although reprogramming of cellular metabolism is a hallmark of cancer, little is known about how metabolic reprogramming contributes to early stages of transformation. Here, we show that the histone deacetylase SIRT6 regulates tumor initiation during intestinal cancer by controlling glucose metabolism. Loss of SIRT6 results in an increase in the number of intestinal stem cells (ISCs), which translates into enhanced tumor initiating potential in APCmin mice. By tracking down the connection between glucose metabolism and tumor initiation, we find a metabolic compartmentalization within the intestinal epithelium and adenomas, where a rare population of cells exhibit features of Warburg-like metabolism characterized by high pyruvate dehydrogenase kinase (PDK) activity. Our results show that these cells are quiescent cells expressing +4 ISCs and enteroendocrine markers. Active glycolysis in these cells suppresses ROS accumulation and enhances their stem cell and tumorigenic potential. Our studies reveal that aerobic glycolysis represents a heterogeneous feature of cancer, and indicate that this metabolic adaptation can occur in non-dividing cells, suggesting a role for the Warburg effect beyond biomass production in tumors.

IgG4-related disease is characterised by the overexpression of immunomodulatory proteins.

AIMS: Immunoglobulin 4-related disease (IgG4-RD) is a multisystem disease, characterised by tumefactive lesions and a swift response to immunosuppressive therapy. Although elevated serum and tissue IgG4 are characteristic, T cells appear to be the primary driver of this immunologically mediated disease. The overarching goal was to examine the role of immunomodulatory cells in IgG4-RD. METHODS AND RESULTS: Biopsies from patients with IgG4-RD (n = 39) and mimics of this disease (n = 78) were evaluated for IgG4, IgG, CD8, programmed cell death ligand 1 (PD-L1) and a subset (n = 18) evaluated for CD4, purine rich box 1 (PU.1), forkhead box protein 3 (FoxP3), PD-L1, programmed cell death 1 (PD-1), indoleamine 2,3-dioxygenase 1 (IDO1) and lymphocyte-activation gene 3 (LAG3). Data pertaining to demographics and laboratory findings at baseline evaluation was extracted from electronic medical records. When compared to mimics, IgG4-RD showed increased numbers of PD-L1- (P = 0.0001), PD-1- (P = 0.001), IDO1- (P = 0.03), LAG3- (P = 0.04) and FoxP3- (P = 0.04)-positive immune cells. The PD-L1-positive cells were enriched within aggregates of CD4 and CD8-positive T cells. Thirty-one of 39 (80%) IgG4-RD cases showed greater than five PD-L1-positive cells per high-power field (HPF), while four of 78 (5%) mimics of this disease exceeded this cut-point. In IgG4-RD, PD-L1-positive macrophages correlated with PD-1- (P = 0.002), LAG3- (P = 0.001) and IDO1-positive cells (P = 0.001); a-positive correlation was also noted between IgG4/IgG ratio and PD-L1-, PD-1- and IDO1-positive cells. CONCLUSIONS: IgG4-RD shows expansion of mechanisms that maintain peripheral tolerance. The spatial and temporal relationship between T cells and the PD-L1-PD-1 axis and the up-regulation of multiple immunomodulatory proteins suggests that these immunoregulatory mechanisms play a significant role in IgG4-RD.

Comparison of RNA In Situ Hybridization and Immunohistochemistry Techniques for the Detection and Localization of SARS-CoV-2 in Human Tissues.

Coronavirus disease-19 (COVID-19) is caused by a newly discovered coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although SARS-CoV-2 is visualized on electron microscopy, there is an increasing demand for widely applicable techniques to visualize viral components within tissue specimens. Viral protein and RNA can be detected on formalin-fixed paraffin-embedded (FFPE) tissue using immunohistochemistry (IHC) and in situ hybridization (ISH), respectively. Herein, we evaluate the staining performance of ISH for SARS-CoV-2 and an IHC directed at the SARS-CoV nucleocapsid protein and compare these results to a gold standard, tissue quantitative real-time polymerase chain reaction (qRT-PCR). We evaluated FFPE sections from 8 COVID-19 autopsies, including 19 pulmonary and 39 extrapulmonary samples including the heart, liver, kidney, small intestine, skin, adipose tissue, and bone marrow. We performed RNA-ISH for SARS-CoV-2 on all cases with IHC for SARS-CoV and SARS-CoV-2 qRT-PCR performed on selected cases. Lungs from 37 autopsies performed before the COVID-19 pandemic served as negative controls. The ISH and IHC slides were reviewed by 4 observers to record a consensus opinion. Selected ISH and IHC slides were also reviewed by 4 independent observers. Evidence of SARS-CoV-2 was identified on both the IHC and ISH platforms. Within the postmortem lung, detected viral protein and RNA were often extracellular, predominantly within hyaline membranes in patients with diffuse alveolar damage. Among individual cases, there was regional variation in the amount of detectable virus in lung samples. Intracellular viral RNA and protein was localized to pneumocytes and immune cells. Viral RNA was detected on RNA-ISH in 13 of 19 (68%) pulmonary FFPE blocks from patients with COVID-19. Viral protein was detected on IHC in 8 of 9 (88%) pulmonary FFPE blocks from patients with COVID-19, although in 5 cases the stain was interpreted as equivocal. From the control cohort, FFPE blocks from all 37 patients were negative for SARS-CoV-2 RNA-ISH, whereas 5 of 13 cases were positive on IHC. Collectively, when compared with qRT-PCR on individual tissue blocks, the sensitivity and specificity for ISH was 86.7% and 100%, respectively, while those for IHC were 85.7% and 53.3%, respectively. The interobserver variability for ISH ranged from moderate to almost perfect, whereas that for IHC ranged from slight to moderate. All extrapulmonary samples from COVID-19-positive cases were negative for SARS-CoV-2 by ISH, IHC, and qRT-PCR. SARS-CoV-2 is detectable on both RNA-ISH and nucleocapsid IHC. In the lung, viral RNA and nucleocapsid protein is predominantly extracellular and within hyaline membranes in some cases, while intracellular locations are more prominent in others. The intracellular virus is detected within pneumocytes, bronchial epithelial cells, and possibly immune cells. The ISH platform is more specific, easier to analyze and the interpretation is associated with the improved interobserver agreement. ISH, IHC, and qRT-PCR failed to detect the virus in the heart, liver, and kidney.

Temporal and spatial heterogeneity of host response to SARS-CoV-2 pulmonary infection.

The relationship of SARS-CoV-2 pulmonary infection and severity of disease is not fully understood. Here we show analysis of autopsy specimens from 24 patients who succumbed to SARS-CoV-2 infection using a combination of different RNA and protein analytical platforms to characterize inter-patient and intra-patient heterogeneity of pulmonary virus infection. There is a spectrum of high and low virus cases associated with duration of disease. High viral cases have high activation of interferon pathway genes and a predominant M1-like macrophage infiltrate. Low viral cases are more heterogeneous likely reflecting inherent patient differences in the evolution of host response, but there is consistent indication of pulmonary epithelial cell recovery based on napsin A immunohistochemistry and RNA expression of surfactant and mucin genes. Using a digital spatial profiling platform, we find the virus corresponds to distinct spatial expression of interferon response genes demonstrating the intra-pulmonary heterogeneity of SARS-CoV-2 infection.

A Possible Case of Vertical Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a Newborn With Positive Placental In Situ Hybridization of SARS-CoV-2 RNA.

Little is known about the effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the coronavirus disease 2019 (COVID-19) on pregnant mothers and their infants. Moreover, there is no definitive evidence that SARS CoV- 2 can be vertically transmitted from an infected mother to the unborn fetus.

Temporal and Spatial Heterogeneity of Host Response to SARS-CoV-2 Pulmonary Infection.

The relationship of SARS-CoV-2 lung infection and severity of pulmonary disease is not fully understood. We analyzed autopsy specimens from 24 patients who succumbed to SARS-CoV-2 infection using a combination of different RNA and protein analytical platforms to characterize inter- and intra- patient heterogeneity of pulmonary virus infection. There was a spectrum of high and low virus cases that was associated with duration of disease and activation of interferon pathway genes. Using a digital spatial profiling platform, the virus corresponded to distinct spatial expression of interferon response genes and immune checkpoint genes demonstrating the intra-pulmonary heterogeneity of SARS-CoV-2 infection.

SARS-CoV-2 can infect the placenta and is not associated with specific placental histopathology: a series of 19 placentas from COVID-19-positive mothers.

Congenital infection of SARS-CoV-2 appears to be exceptionally rare despite many cases of COVID-19 during pregnancy. Robust proof of placental infection requires demonstration of viral localization within placental tissue. Only two of the few cases of possible vertical transmission have demonstrated placental infection. None have shown placental expression of the ACE2 or TMPRSS2 protein, both required for viral infection. We examined 19 COVID-19 exposed placentas for histopathologic findings, and for expression of ACE2, and TMPRSS2 by immunohistochemistry. Direct placental SARS-CoV-2 expression was studied by two methods-nucleocapsid protein expression by immunohistochemistry, and RNA expression by in situ hybridization. ACE2 membranous expression in the syncytiotrophoblast (ST) of the chorionic villi is predominantly in a polarized pattern with expression highest on the stromal side of the ST. In addition, cytotrophoblast and extravillous trophoblast express ACE2. No ACE2 expression was detected in villous stroma, Hofbauer cells, or endothelial cells. TMPRSS2 expression was only present weakly in the villous endothelium and rarely in the ST. In 2 of 19 cases, SARS-CoV-2 RNA was present in the placenta focally in the ST and cytotrophoblast. There was no characteristic histopathology present in our cases including the two placental infections. We found that the placenta is capable of being infected but that this event is rare. We propose one explanation could be the polarized expression of ACE2 away from the maternal blood and pronounced paucity of TMPRSS2 expression in trophoblast.

Pancreatic circulating tumor cell profiling identifies LIN28B as a metastasis driver and drug target.

Pancreatic ductal adenocarcinoma (PDAC) lethality is due to metastatic dissemination. Characterization of rare, heterogeneous circulating tumor cells (CTCs) can provide insight into metastasis and guide development of novel therapies. Using the CTC-iChip to purify CTCs from PDAC patients for RNA-seq characterization, we identify three major correlated gene sets, with stemness genes LIN28B/KLF4, WNT5A, and LGALS3 enriched in each correlated gene set; only LIN28B CTC expression was prognostic. CRISPR knockout of LIN28B-an oncofetal RNA-binding protein exerting diverse effects via negative regulation of let-7 miRNAs and other RNA targets-in cell and animal models confers a less aggressive/metastatic phenotype. This correlates with de-repression of let-7 miRNAs and is mimicked by silencing of downstream let-7 target HMGA2 or chemical inhibition of LIN28B/let-7 binding. Molecular characterization of CTCs provides a unique opportunity to correlated gene set metastatic profiles, identify drivers of dissemination, and develop therapies targeting the "seeds" of metastasis.

Stromal Microenvironment Shapes the Intratumoral Architecture of Pancreatic Cancer.

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

A tumor-specific endogenous repetitive element is induced by herpesviruses.

Tandem satellite repeats account for 3% of the human genome. One of them, Human Satellite II (HSATII), is highly expressed in several epithelial cancers and cancer cell lines. Here we report an acute induction of HSATII RNA in human cells infected with two herpes viruses. We show that human cytomegalovirus (HCMV) IE1 and IE2 proteins cooperate to induce HSATII RNA affecting several aspects of the HCMV replication cycle, viral titers and infected-cell processes. HSATII RNA expression in tissue from two chronic HCMV colitis patients correlates with the strength of CMV antigen staining. Thus, endogenous HSATII RNA synthesis after herpesvirus infections appears to have functionally important consequences for viral replication and may provide a novel insight into viral pathogenesis. The HSATII induction seen in both infected and cancer cells suggests possible convergence upon common HSATII-based regulatory mechanisms in these seemingly disparate diseases.

Improved Detection of Circulating Epithelial Cells in Patients with Intraductal Papillary Mucinous Neoplasms.

BACKGROUND: Recent work has demonstrated early shedding of circulating epithelial cells (CECs) from premalignant intraductal papillary mucinous neoplasms (IPMNs). However, the potential use of CECs as a "liquid biopsy" for patients with IPMNs has been limited by antigen dependence of CEC isolation devices and the lack of robust detection biomarkers across CEC phenotypes. MATERIALS AND METHODS: We utilized a negative depletion microfluidic platform to purify CECs from contaminating leukocytes and coupled this platform with immunofluorescence, RNA in situ hybridization, and RNA sequencing (RNA-seq) detection and enumeration. RESULTS: Using established protein (EpCAM, cytokeratins) and novel noncoding RNA (HSATII, cytokeratins) biomarkers, we detected CECs in 88% of patients bearing IPMN lesions. RNA-seq analysis for MUC genes confirm the likely origin of these CECs from pancreatic lesions. CONCLUSION: Our findings increase the sensitivity of detection of these cells and therefore could have clinical implications for cancer risk stratification. IMPLICATIONS FOR PRACTICE: This work describes a high-sensitivity platform for detection of epithelial cells shed from preneoplastic lesions at high risk of malignant transformation. Further research efforts are underway to define the transcriptional programs that might allow discrimination between circulating cells released from tumors that will become malignant and cells released from tumors that will not. After further refinement, this combination of technologies could be deployed for monitoring and early detection of patients at high risk for developing new or recurrent pancreatic malignancies.

Diverse repetitive element RNA expression defines epigenetic and immunologic features of colon cancer.

There is tremendous excitement for the potential of epigenetic therapies in cancer, but the ability to predict and monitor response to these drugs remains elusive. This is in part due to the inability to differentiate the direct cytotoxic and the immunomodulatory effects of these drugs. The DNA-hypomethylating agent 5-azacitidine (AZA) has shown these distinct effects in colon cancer and appears to be linked to the derepression of repeat RNAs. LINE and HERV are two of the largest classes of repeats in the genome, and despite many commonalities, we found that there is heterogeneity in behavior among repeat subtypes. Specifically, the LINE-1 and HERV-H subtypes detected by RNA sequencing and RNA in situ hybridization in colon cancers had distinct expression patterns, which suggested that these repeats are correlated to transcriptional programs marking different biological states. We found that low LINE-1 expression correlates with global DNA hypermethylation, wild-type TP53 status, and responsiveness to AZA. HERV-H repeats were not concordant with LINE-1 expression but were found to be linked with differences in FOXP3+ Treg tumor infiltrates. Together, distinct repeat RNA expression patterns define new molecular classifications of colon cancer and provide biomarkers that better distinguish cytotoxic from immunomodulatory effects by epigenetic drugs.