A single tertiary institution review of the international system for serous fluid cytopathology and the impact of cell block and ancillary studies on its performance.

INTRODUCTION: We sought to assess the utility of the International System for Serous Fluid Cytopathology (TIS) in the context of our department's routine practice. MATERIALS AND METHODS: We examined 1028 archived effusion cytology (pleural, peritoneal, and pericardial) cases from 2018 to 2019, and re-classified them along the international system into the following diagnostic categories: nondiagnostic (ND), negative for malignancy (NFM), atypia cells of undetermined significance (AUS), suspicious for malignancy (SFM), and malignant (MAL). RESULTS: The full distribution of the cases examined was as follows: ND 2.0%; NFM 66.1%; AUS 6.0%; SFM 4.7%; MAL 21.2%. Overall risk of malignancy for each category was calculated as: ND 30.0%; NFM 18.0%; AUS 61.9%; SFM 100%; MAL 94.4%. The overall performance attributes of TIS were as follows: sensitivity 57.1%; specificity 98.3%; positive predictive value 94.4%; negative predictive value 82.0%; diagnostic accuracy 84.5%. CONCLUSIONS: The new classification was simple and intuitive to use and our results appear to fall within the expected ranges of the new guidelines, with risk of malignancy and accuracy comparable to similar studies. The availability of a cell block allowed for refinement of the diagnosis in a majority of cases with equivocal cytology, though this was dependent on the cell yield.

Epithelial-mesenchymal transition and cancer stem cell interactions in breast phyllodes tumours: immunohistochemical evaluation of EZH2, EZR, HMGA2, CD24 and CD44 in correlation with outcome analysis.

AIM: Phyllodes tumours (PTs) categorised as benign, borderline and malignant, account for 1% of all breast tumours. Histological assessment does not always predict tumour behaviour, hindering determination of the clinical course and management.Epithelial-mesenchymal transition (EMT) is an important process during embryogenesis. Dysregulation of EMT causes loss of cell polarity, decreased intercellular adhesion, increased motility and invasiveness, promoting tumour progression. Similarly, cancer stem cells (CSCs) promote tumour growth, resistance and recurrence. The aim of this study is to evaluate expression of CSC markers; enhancer of zeste homolog 2 (EZH2), CD24 and CD44 and EMT associated proteins; ezrin (EZR) and high-mobility group AT-hook 2 (HMGA2) in PTs. METHOD: Uing tissue microarray sections, immunohistochemistry was performed on 360 PTs. Epithelial and stromal expressions of EZH2, EZR, HMGA2, CD24 and CD44 were evaluated to assess their impact on disease progression and behaviour in correlation with clinicopathological parameters. RESULTS: Stromal expression of EZH2, EZR and HMGA2 was observed in 73 (20.3%), 53 (14.7%) and 28 (7.8%) of tumours, epithelial expression in 121 (35.9%), 3 (0.8%) and 351 (97.5%) tumours, respectively. CD24 and CD44 staining was absent in both components. CONCLUSION: Expression of biomarkers correlated significantly with aggressive tumour traits such as stromal hypercellularity, atypia, mitoses and permeative tumour borders.Stromal expression of EZH2 and EZR shortened disease-free survival and overall survival; HMGA2 expression did not alter patient survival. EZH2 and EZR may thus be useful in predicting PT behaviour.

Microwave assisted one-pot synthesis, photophysical and physicochemical studies of novel biologically active heterocyclic Donor (D)-π-Acceptor (A) chromophore.

A donor-π-acceptor (D-π-A) chromophore, 2-amino-4-(9-ethyl-9H-carbazol-3-yl)-8-methoxy-5,6-dihydrobenzo[h]quinoline-3-carbonitrile (AEDQ) was synthesized from the condensation of 6-methoxy-3,4-dihydronaphthalen-1(2H)-one, 9-ethyl-9H-carbazole-3-carbaldehyde, malononitrile and NH4OAc in ethanol. Spectroscopic techniques and elemental analysis were employed to establish the structure of AEDQ. Photophysical parameters and fluorescence quantum yield were calculated in the different polarity solvents to evaluate the interactions of the solvent with AEDQ chromophore. Further, the interaction of the AEDQ with cationic and anionic surfactants (CTAB, SDS) were also evaluated by using fluorescence spectroscopy techniques. The intensity of the fluorescence spectrum increased as the concentration of surfactants increased, suggesting that strong interaction occurs between AEDQ with surfactants, and this interaction arises from electrostatic forces. As a result, the AEDQ chromophore could be used to determine the CMC of surfactants. The disc diffusion and minimal inhibitory concentration (MIC) technique were used to test in-vitro antibacterial activity against Gram +ve and Gram -ve bacteria, and the results are compared with the standard drug, tetracycline. AEDQ also showed good ADMET, pharmacokinetics and drug-likeness properties, which are desirable for a good drug candidate. The molecule also fits well in the DNA gyrase A active pocket site with the binding free energy of -17.92 kcal/mol, which testifies its good antibacterial activity.

Closure of a Recurrent Urethrovaginal Fistula in a Girl with Cloacal Anomaly Using Deflux Injection.

In a girl born with cloaca, both hemivaginae and rectum were located above the bladder neck, and both ureters were connected to the hemivaginae. After diverting colostomy and cystovaginoscopy on the second day of life, the repair of cloaca was performed at 10 months of age by posterior sagittal anorecto vaginoplasty (PSARVP), including laparotomy and bilateral ureteric reimplantation. Eight months after the surgery, she developed a vesicovaginal fistula, which was repaired and closed by open surgery through the bladder. Three months after this procedure, a tiny urethrovaginal fistula was noticed, which was closed at the age of 2 years using hook diathermy to refresh the edges and was then closed by Deflux injection. The proper closure of the urethrovaginal fistula was confirmed by radiology and cystoscopy 3 months after the surgery. This report shows that injection of Deflux into a tiny urethrovaginal fistula following refreshing the edges may be a valid treatment option in selected cases.

An automated staining protocol for seven-colour immunofluorescence of human tissue sections for diagnostic and prognostic use.

Multiplex immunofluorescence (mIF) allows simultaneous antibody-based detection and quantification of the expression of up to six markers, plus a nuclear counterstain, on a single tissue section. Recent studies have shown the potential for mIF to advance our understanding of complex disease processes, including cancer. It is important that the technique be standardised and validated to facilitate its transition into clinical use. Traditional approaches to mIF rely on manual processing of sections, which is time-consuming and a source of significant variation between samples/individuals. Here we determined if an automated diagnostic tissue stainer could be used for mIF incorporating tyramide signal amplification (TSA), and how the final image quality compared with sections stained semi-automatically or manually. Using tissue microarrays of fixed human breast tumour sections, we observed comparable antibody labelling between the diagnostic autostainer and manual technique. The diagnostic autostainer produced higher signal intensity with similar spectral unmixing efficiency. We also found that microwave treatment for antibody stripping during TSA labelling could be replaced by the heating option incorporated within the diagnostic-use autostainer. These data show that diagnostic autostainers used for traditional immunohistochemistry protocols can be readily adapted to achieve rapid preparation of high-quality sections using a TSA method for clinical mIF.

Sgs1 Binding to Rad51 Stimulates Homology-Directed DNA Repair in Saccharomyces cerevisiae.

Accurate repair of DNA breaks is essential to maintain genome integrity and cellular fitness. Sgs1, the sole member of the RecQ family of DNA helicases in Saccharomyces cerevisiae, is important for both early and late stages of homology-dependent repair. Its large number of physical and genetic interactions with DNA recombination, repair, and replication factors has established Sgs1 as a key player in the maintenance of genome integrity. To determine the significance of Sgs1 binding to the strand-exchange factor Rad51, we have identified a single amino acid change at the C-terminal of the helicase core of Sgs1 that disrupts Rad51 binding. In contrast to an SGS1 deletion or a helicase-defective sgs1 allele, this new separation-of-function allele, sgs1-FD, does not cause DNA damage hypersensitivity or genome instability, but exhibits negative and positive genetic interactions with sae2Δ, mre11Δ, exo1Δ, srs2Δ, rrm3Δ, and pol32Δ that are distinct from those of known sgs1 mutants. Our findings suggest that the Sgs1-Rad51 interaction stimulates homologous recombination (HR). However, unlike sgs1 mutations, which impair the resection of DNA double-strand ends, negative genetic interactions of the sgs1-FD allele are not suppressed by YKU70 deletion. We propose that the Sgs1-Rad51 interaction stimulates HR by facilitating the formation of the presynaptic Rad51 filament, possibly by Sgs1 competing with single-stranded DNA for replication protein A binding during resection.

Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation.

Reversible detyrosination of α-tubulin is crucial to microtubule dynamics and functions, and defects have been implicated in cancer, brain disorganization, and cardiomyopathies. The identity of the tubulin tyrosine carboxypeptidase (TCP) responsible for detyrosination has remained unclear. We used chemical proteomics with a potent irreversible inhibitor to show that the major brain TCP is a complex of vasohibin-1 (VASH1) with the small vasohibin binding protein (SVBP). VASH1 and its homolog VASH2, when complexed with SVBP, exhibited robust and specific Tyr/Phe carboxypeptidase activity on microtubules. Knockdown of vasohibins or SVBP and/or inhibitor addition in cultured neurons reduced detyrosinated α-tubulin levels and caused severe differentiation defects. Furthermore, knockdown of vasohibins disrupted neuronal migration in developing mouse neocortex. Thus, vasohibin/SVBP complexes represent long-sought TCP enzymes.

Clinicopathological characteristics of oestrogen receptor negative, progesterone receptor positive breast cancers: re-evaluating subsets within this group.

AIMS: The presence of oestrogen and progesterone receptors (ER, PR) in breast carcinoma is an important prognostic indicator as well as a predictor of likely response to hormonal treatment. Current ambiguity surrounds ER-negative (-)/PR-positive (+) breast cancer (BC) as to whether this phenotype exists as a distinct entity. The independent predictive value of PR for treatment considerations is also in question, as some investigators believe ER status to be the single most important therapeutic predictive factor in BC. We undertook this study to determine the existence of ER(-)/PR(+) BC and the prognostic effect, if any, of this phenotype. METHODS: We investigated 267 archival documented ER(-)/PR(+) BCs diagnosed between January 1994 and July 2009. Histological slides were retrieved and reviewed. Tissue microarrays were constructed by selecting two 1 mm cores of tumour per case. Repeat immunohistochemistry was performed for confirmation of the ER(-)/PR(+) status. Clinicopathological parameters including age, ethnicity, tumour size, histological grade, histological subtype, associated ductal carcinoma in situ, lymphovascular invasion and lymph node status were evaluated. RESULTS: On repeat immunohistochemistry, 92 tumours were confirmed as ER(-)/PR(+) BCs. This phenotype accounted for 1.1% of all BC phenotypes and exhibited different clinicopathological features and survival outcome when compared with other phenotypes. ER(-)/PR(+) tumours showed a trend for an early recurrence and poorer overall survival as compared with the patients with ER(+)/PR(+) tumours and similar to ER(-)/PR(-) tumours. CONCLUSIONS: Our findings suggest that ER(-)/PR(+) BCs exist, although rare, with distinct pathological and clinical characteristics from patients with ER(+)/PR(+) BCs.

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression.

In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186-212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks.

A case of small bowel metastasis from spinal Ewing sarcoma causing intussusception in an adult female.

BACKGROUND: Ewing sarcomas are highly aggressive malignant tumours occurring predominantly in the long bones of the extremities in children and young adults. About 20 % of patients will present with metastases at diagnosis with the commonest sites being the lungs, bone and bone marrow. Cases of primary small bowel Ewing sarcomas have been described but are nonetheless exceedingly rare, even more so cases of metastasis to the small bowel. CASE PRESENTATION: We describe a case of vertebral Ewing sarcoma in a 44 year-old female which metastasized to the jejunum causing intussusception. CONCLUSIONS: Ewing's sarcoma is highly aggressive and presence of metastases, overt or subclinical, is thought to be present in almost all patients at diagnosis. As evidenced by our patient, metastatic disease can progress rapidly to cause further complications and confer a poorer survival. The possibility of metastasis, no matter how rare or unlikely the site is, should be considered and actively investigated to expedite treatment of the primary disease.

Structural Motifs Critical for In Vivo Function and Stability of the RecQ-Mediated Genome Instability Protein Rmi1.

Rmi1 is a member of the Sgs1/Top3/Rmi1 (STR) complex of Saccharomyces cerevisiae and has been implicated in binding and catalytic enhancement of Top3 in the dissolution of double Holliday junctions. Deletion of RMI1 results in a severe growth defect resembling that of top3Δ. Despite the importance of Rmi1 for cell viability, little is known about its functional domains, particularly in Rmi1 of S. cerevisiae, which does not have a resolved crystal structure and the primary sequence is poorly conserved. Here, we rationally designed point mutations based on bioinformatics analysis of order/disorder and helical propensity to define three functionally important motifs in yeast Rmi1 outside of the proposed OB-fold core. Replacing residues F63, Y218 and E220 with proline, designed to break predicted N-terminal and C-terminal α-helices, or with lysine, designed to eliminate hydrophobic residues at positions 63 and 218, while maintaining α-helical structure, caused hypersensitivity to hydroxyurea. Further, Y218P and E220P mutations, but not F63P and F63K mutations, led to reduced Rmi1 levels compared to wild type Rmi1, suggesting a role of the C-terminal α-helix in Rmi1 stabilization, most likely by protecting the integrity of the OB-fold core. Our bioinformatics analysis also suggests the presence of a disordered linker between the N-terminal α-helix and the OB fold core; a P88A mutation, designed to increase helicity in this linker, also impaired Rmi1 function in vivo. In conclusion, we propose a model that maps all functionally important structural features for yeast Rmi1 based on biological findings in yeast and structure-prediction-based alignment with the recently established crystal structure of the N-terminus of human Rmi1.

Sentinel lymph nodes with isolated tumour cells and micrometastases in breast cancer: clinical relevance and prognostic significance.

AIM: We performed a retrospective review to determine the prognostic significance of isolated tumour cells (ITCs) and micrometastases to the sentinel lymph nodes of patients with breast cancer. METHODS: A total of 1044 patients with a diagnosis of invasive carcinoma of the breast who underwent surgical treatment including the sentinel lymph node biopsy procedure from July 2004 to October 2009 were included in the study. RESULTS: In 710 (68%) patients, no metastasis was seen to the sentinel lymph nodes. ITCs were detected in 22 (2.1%) patients, micrometastasis in 52 (5.0%) and macrometastases in 260 (24.9%). With a median follow-up of 28.8 months, disease recurrence was seen in 38 (3.6%) patients and 15 (1.5%) patients died of disease. No disease recurrence or deaths were recorded in women with ITCs in sentinel lymph nodes. In the micrometastasis group, 2 patients suffered disease recurrence and both died of disease. CONCLUSIONS: We conclude that ITCs in the sentinel lymph nodes did not adversely impact disease free and overall survivals. Although only 2 recurrences with subsequent death occurred in the micrometastasis group, it may suggest a propensity for presence of micrometastases to augur a worse outcome, and justifies continued segregation of ITCs from micrometastasis.

Predicting the likelihood of additional lymph node metastasis in sentinel lymph node positive breast cancer: validation of the Memorial Sloan-Kettering Cancer Centre (MSKCC) nomogram.

AIM: To identify important clinicopathological parameters that are most helpful in predicting additional non-sentinel lymph node (SLN) metastasis among patients with a positive SLN biopsy in the Singapore breast cancer population. METHODS: A total of 1409 patients who underwent SLN biopsy were reviewed over a 5 year period from July 2004 to October 2009. A Singapore General Hospital (SGH) nomogram was developed from predictors in the Memorial Sloan-Kettering Cancer Centre (MSKCC) nomogram using 266 patients with primary invasive breast cancer and a positive SLN biopsy who subsequently had an axillary lymph node dissection. The SGH nomogram was calibrated using bootstrapped data, while the MSKCC nomogram was calibrated using SGH data. The performance of these two nomograms was compared with the calculation of the area under the receiver-operator characteristics curve and adequacy indices. RESULTS: The MSKCC nomogram achieved an area under the curve (AUC) of 0.716 (range 0.653-0.779) in our study population, while the SGH nomogram, which used only three pathological parameters, lymphovascular invasion, number of positive and negative SLN biopsies, achieved an AUC of 0.750 (range 0.691-0.808). The SGH nomogram with a higher adequacy index (0.969) provided better estimates compared with the MSKCC nomogram (0.689). CONCLUSIONS: The use of the MSKCC nomogram was validated in our local patient population. The SGH nomogram showed promise to be equally, if not, more predictive as a model in our own population, while using only three pathological parameters.

Improved quenched fluorescent probe for imaging of cysteine cathepsin activity.

The cysteine cathepsins are a family of proteases that play important roles in both normal cellular physiology and many human diseases. In cancer, the activity of many of the cysteine cathepsins is upregulated and can be exploited for tumor imaging. Here we present the design and synthesis of a new class of quenched fluorescent activity-based probes (qABPs) containing a phenoxymethyl ketone (PMK) electrophile. These reagents show enhanced in vivo properties and broad reactivity resulting in dramatically improved labeling and tumor imaging properties compared to those of previously reported ABPs.

Coupling protein engineering with probe design to inhibit and image matrix metalloproteinases with controlled specificity.

Matrix metalloproteinases (MMPs) are zinc endopeptidases that play roles in numerous pathophysiological processes and therefore are promising drug targets. However, the large size of this family and a lack of highly selective compounds that can be used for imaging or inhibition of specific MMPs members has limited efforts to better define their biological function. Here we describe a protein engineering strategy coupled with small-molecule probe design to selectively target individual members of the MMP family. Specifically, we introduce a cysteine residue near the active-site of a selected protease that does not alter its overall activity or function but allows direct covalent modification by a small-molecule probe containing a reactive electrophile. This specific engineered interaction between the probe and the target protease provides a means to both image and inhibit the modified protease with absolute specificity. Here we demonstrate the feasibility of the approach for two distinct MMP proteases, MMP-12 and MT1-MMP (or MMP-14).

Functional imaging of legumain in cancer using a new quenched activity-based probe.

Legumain is a lysosomal cysteine protease whose biological function remains poorly defined. Legumain activity is up-regulated in most human cancers and inflammatory diseases most likely as the result of high expression in populations of activated macrophages. Within the tumor microenvironment, legumain activity is thought to promote tumorigenesis. To obtain a greater understanding of the role of legumain activity during cancer progression and inflammation, we developed an activity-based probe that becomes fluorescent only upon binding active legumain. This probe is highly selective for legumain, even in the context of whole cells and tissues, and is also a more effective label of legumain than previously reported probes. Here we present the synthesis and application of our probe to the analysis of legumain activity in primary macrophages and in two mouse models of cancer. We find that legumain activity is highly correlated with macrophage activation and furthermore that it is an ideal marker for primary tumor inflammation and early stage metastatic lesions.

Non-invasive imaging of cysteine cathepsin activity in solid tumors using a 64Cu-labeled activity-based probe.

The papain family of cysteine cathepsins are actively involved in multiple stages of tumorigenesis. Because elevated cathepsin activity can be found in many types of human cancers, they are promising biomarkers that can be used to target radiological contrast agents for tumor detection. However, currently there are no radiological imaging agents available for these important molecular targets. We report here the development of positron emission tomography (PET) radionuclide-labeled probes that target the cysteine cathepsins by formation of an enzyme activity-dependent bond with the active site cysteine. These probes contain an acyloxymethyl ketone (AOMK) functional group that irreversibly labels the active site cysteine of papain family proteases attached to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) tag for labeling with (64)Cu for PET imaging studies. We performed biodistribution and microPET imaging studies in nude mice bearing subcutaneous tumors expressing various levels of cysteine cathepsin activity and found that the extent of probe uptake by tumors correlated with overall protease activity as measured by biochemical methods. Furthermore, probe signals could be reduced by pre-treatment with a general cathepsin inhibitor. We also found that inclusion of a Cy5 tag on the probe increased tumor uptake relative to probes lacking this fluorogenic dye. Overall, these results demonstrate that small molecule activity-based probes carrying radio-tracers can be used to image protease activity in living subjects.

Five-substrate cocktail as a sensor array for measuring enzyme activity fingerprints of lipases and esterases.

Substrate arrays for measuring enzyme activity fingerprints can be conveniently formulated as cocktails designed such that the reaction products can be separated and quantified by analytical high-performance liquid chromatography (HPLC). Fingerprinting of lipases and esterases, an important class of microbial enzymes, is reported with a cocktail of only five substrates as a practical fingerprinting reagent. An unusually strong C4-esterase activity was thus revealed in a recently discovered microbial esterase.

HER2/neu revisited: quality and interpretive issues.

BACKGROUND: Immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) are the only tests currently approved by the US Food and Drug Administration for classifying which patients will benefit from trastuzumab therapy. The accuracy of these two testing methods can be adversely affected by a variety of pre-analytical, analytical and post-analytical factors. According to the latest published recommendations of the panel of the Joint Committee of the American Society of Clinical Oncology and College of American Pathologists for HER2/neu testing, laboratories performing IHC and FISH for HER2/neu status in breast cancer are now required to have a high concordance of at least 95% between IHC and FISH, significantly higher than that in the published literature. AIM: To perform a retrospective analysis of the concordance of IHC and FISH analysis for HER2/neu at Singapore General Hospital and review potential causes of disparity between these two methods. METHOD: A retrospective review of a total of 106 invasive ductal carcinomas evaluated for HER2/neu at the Department of Pathology, Singapore General Hospital between 2007 and 2008 were included in the study. The initial HER2/neu immunostained slides were reviewed independently without knowledge of FISH results, and concordance between IHC and FISH was determined. RESULTS: Concordance between IHC and FISH assay was excellent and within the published range (104/106=98.1%). The discordant cases represent a well-recognised subset of genetic heterogeneity in HER2/neu, which is known to contribute to positive IHC and negative FISH tests.