Enhancement of gold-curcumin nanoparticle mediated radiation response for improved therapy in cervical cancer: a computational approach and predictive pathway analysis.

Radiotherapy is prevalently applied for highly effective cancer therapy while the low specificity of radiation is deleterious to the nearby healthy cells. High-Z-based nanomaterials offer excellent radio-enhancement properties while natural products provide radioprotection. Modulation of the radiotherapeutic index via applying nanomaterials is feasible for effective treatment however, the scenario changes when simultaneous protection of non-cancerous cells is required. Here, we report the modulatory radiotherapeutic effect of curcumin conjugated gold nanoparticles in a single nanoformulation to pave the long-awaited hope of a single combination-based, cell-selective radio enhancer, and protectant for cancer radiotherapy. We have validated the effective radiation dose along with the combination of the radio-nano-modulator by a reverse experimentation statistical model. The concept was supported by different sets of experiments, like quantification of ROS generation, cell cycle monitoring, mitochondrial membrane potential measurement, etc. along with gene expression study, and predictive modeling of molecular pathways of the killing mechanism. In conclusion, the nanoconjugate showed a promise to become a candidate for the pH-dependent cell-specific radio-modulator.

Reproducibility of the "International Academy of Cytology Yokohama System for Reporting Breast Cytology" - A Retrospective Analysis of 70 Cases.

Background: Fine-needle aspiration cytology (FNAC) is the most practiced initial method for evaluation of breast lesions. The International Academy of Cytology Yokohama System for Reporting Breast (IAC YSRB) Fine-Needle Aspiration Biopsy Cytopathology has been developed to standardize the reporting system. However, literature available on the inter-observer reproducibility of 5 IAC YSRB categories is limited. Aim: We investigated the inter-observer reproducibility of the IAC YSRB system. Method and Materials: A total of 70 consecutive specimens obtained from FNAC of breast lesions were reviewed retrospectively by 3 experienced cytopathologists who allotted 1 to 5 IAC YSRB categories. Results: The percent overall agreement between observers was 70.48% and the free marginal kappa was 0.63, which signifies substantial agreement. After combining "Suspicious" and "Malignant" categories, overall agreement was 80.95% and free marginal kappa became 0.75. Conclusions: Inter-observer agreement of three (70.48%) cytopathologists was substantial. Agreement can be improved by combining certain categories, especially "Suspicious" and "Malignant". Technical quality limitation plays a significant role in a proportion of cases, mainly the "Atypical" and "Suspicious of malignancy" categories. Application of the IAC YSRB system in day-to-day practice will increase the inter-observer agreement.

A study of CD10 positivity of stromal cells in core needle biopsy specimen of breast cancer patients and its relation with histological grade and lymphovascular invasion.

BACKGROUND: In core needle biopsy (CNB) often the histological grade of invasive breast carcinoma is under-estimated due to heterogeneity of epithelial component. Stroma is relatively homogenous throughout the tumor and strong CD10 stromal positivity is proposed to be associated with high tumor grade. AIMS AND OBJECTIVES: The aim of this work was to study the expression of CD10 in stromal cells of invasive carcinoma of breast, no specific type (NST) in CNB specimens, and analyze its association with final histological grade and lymphovascular invasion (LVI). MATERIALS AND METHODS: A total of 50 cases of invasive carcinoma of breast, NST were studied for 18 months. CNB specimens were graded according to modified Scarff-Bloom-Richardson (SBR) system and CD10 positivity was assessed in stromal cells. Mastectomy specimens were also similarly graded. Relation of stromal CD10 positivity with histological grading and LVI was studied. STATISTICS: Associations between the variables were studied by Chi-square test. A value of P < 0.05 was considered to be statistically significant. RESULTS: On CNB 46% patients had a grade 2 tumor, followed by 30% grade 3 and 24% grade 1 tumor. Strong CD10 positivity was seen in 40% cases, 32% showed weak positivity and 28% were negative for CD10 in stromal cells in CNB specimen. On evaluation of mastectomy specimen 48% of the patients had a grade 2 tumor, followed by 40% grade 3 tumor and 12% grade 1 tumor. Strong CD10 positivity was found to be significantly associated with final grade 3 tumor (P < 0.001) and LVI (P = 0.005). CONCLUSIONS: There was underestimation of histological grade on CNB, while strong stromal CD10 positivity in CNB was significantly associated with final grade 3 tumor and LVI.

Toll-Like Receptor 9 Expression Levels in Breast Carcinoma Correlate with Improved Overall Survival in Patients Treated with Neoadjuvant Chemotherapy and Could Serve as a Prognostic Marker.

Aim: Toll-like receptor 9 (TLR9) can recognize the DNA fragments released from chemotherapy-treated cancer cells in tumor tissues and induce an inflammatory response. The aim of the present study was to evaluate the survivability benefit of TLR9 expression levels as a potential prognostic marker in breast cancer patients treated with neoadjuvant chemotherapy (NACT). Methods: To study the expression of TLR9 in breast tumor, immunohistochemical (IHC) analyses were performed on two patient cohorts, with NACT (n = 19) and without NACT (n = 23). To corroborate the findings from the in-house cohort, we also used publicly available datasets including SurvExpress (GSE 20685) and the Kaplan-Meier plotter tool (GSE 16446) to analyze the relationship between the expression of TLR9 and overall survivability for NACT. Results: The IHC analyses of our inhouse cohort demonstrated that TLR9 was expressed in both malignant breast epithelial cancer cells as well as in the adjacent stromal cells. The IHC results also indicated that, the percentage of malignant epithelial cells (54.76%) expressing TLR9 was higher than in the adjacent stromal compartment (11.9%). We also observed an increase in the expression levels of TLR9 in the patients who were given NACT (p = 0.0379). Further, the analysis of publicly available datasets demonstrated that elevated TLR9 expression was related to increased overall survival in patients treated with NACT. Conclusions: In this study, we show for the first time that elevated TLR9 tissue expression levels in breast cancer may serve as a prognostic marker for patients treated with NACT and could potentially be used to select the neoadjuvant regime.

Appreciation of Pattern in Diagnosis of Lung Adenocarcinoma from Cytology Specimen: Our Experience with Fine Needle Aspiration Cytology and Cell Block in a Resource Constraint Setup.

BACKGROUND AND AIMS: Advent of personalised treatment needs correct diagnosis of lung adenocarcinoma with its molecular subtyping. Minimal use of special stain or immunohistochemistry (IHC) in small specimens save material for molecular testing. Various histologic patterns in adenocarcinoma (ADC) subtypes have different prognostic implications and current recommendation is to describe these patterns in small specimens. Aim of this study was to diagnose adenocarcinoma from cytology specimens depending on adenocarcinoma pattern on fine needle aspiration smears and cell blocks. We also studied the additional role of cell blocks as a platform for special stain and IHC. MATERIALS AND METHODS: Conventional smears and cell block (CB) preparation were examined from transthoracic CT guided FNA samples of suspicious lung malignancy cases. Clear defining architectural pattern and cytomorphological features in favour of adenocarcinoma were evaluated and mucin stain and IHC were used as and when required. RESULTS: A total of 86 cases were included in this study, of which 83 cases were diagnosed as adenocarcinoma, 52 (62.5%) showed clear cut evidence of adenocarcinoma from smears and CBs. CB morphology alone aided the diagnosis in 12. Various ADC patterns in combination or alone were appreciated in these 64 cases. Sixteen needed mucin stain and 3 needed IHC for diagnosis. Forty one were ADC with solid pattern of which 39 showed high nuclear grade. CONCLUSION: Adequately cellular FNA smears and corresponding cell blocks of optimal quality can aid effectively in diagnosing adenocarcinoma and appreciating its pattern. Therefore, it would minimize the need for special stain and/or IHC with preservation of more material for molecular testing.

Tumor Cell Representation by an Improvised Technique of Fine-Needle Aspiration Specimen Acquisition and Cell Block Preparation: Our Experience in Lung Cancer Cases in a Peripheral Center of Eastern India.

BACKGROUND: Being a minimally invasive diagnostic technique, Fine-Needle Aspiration Cytology (FNAC) has become the first-line test and corresponding aspirated material has become the target specimen for diagnosis and ancillary tests in lung carcinoma. Although the role of Cell Blocks (CBs) in diagnosis and in ancillary testing is well recognized in literature, limited attention has been paid to specimen procurement and triage in the preparation of CBs. In the present scenario, CBs are not consistently optimal because of its low cellularity. AIMS: This study is aimed to describe an improvised technique of specimen acquisition and cell block preparation in CT-guided FNACs of lung carcinoma cases in a resource-constrained center and to assess its efficacy for optimal representation of cellularity, morphology, and architecture. MATERIALS AND METHODS: Total 85 lung carcinoma cases undergoing CT-guided FNAC in our center from February 2017 to January 2018 were included in this study. 4 to 5 direct smears and subsequent CBs were made from material obtained by single pass. Cellularity of smears and corresponding cell blocks were assessed and categorized according to a scoring system (score 1 to 3 for number of cells <50, 50-100, >100, respectively). Preserved architecture and morphology were also assessed in smears and CBs. RESULTS: The evaluated samples showed a cellularity score 3 in 65.4%CBs and score 2 in 24.7% CBs. Overall, 90.1% cell blocks had acceptable cellularity. Cell morphology was preserved in all CBs of acceptable cellularity, except for two adenocarcinoma, one squamous cell carcinoma, and one small cell carcinoma blocks. Cellular architecture was also preserved in all CBs of acceptable cellularity. CONCLUSIONS: This simple improvised technique of CB preparation optimized its cellularity, morphology, and architectural preservation, even after adequate cellular FNA smears.

Reproducibility of "the Papanicolaou Society of Cytopathology system for reporting respiratory cytology" - A retrospective analysis of 101 cases of CT-guided FNAC.

INTRODUCTION: Guided fine-needle aspiration cytology is a popular investigative procedure in diagnosing pulmonary lesions. The Papanicolaou Society of Cytopathology (PSC) has already outlined a categorical system for reporting respiratory cytology. Though each category has a known malignancy risk, their inter observer reproducibility have not been well documented. This study was directed towards establishing the reproducibility of this categorical system in diagnosing pulmonary lesions. METHOD: One hundred and one consecutive cytology specimens obtained by CT-guided FNA from lung lesions were independently reviewed by 3 experienced cytopathologists, who allotted each case to 1 of 6 PSC categories. Statistical analysis for percent overall agreement was done using Fleiss' Kappa. RESULT: Percent overall agreement was 71.29% and free marginal kappa was 0.66. On combining categories "suspicious" and "malignant" percent overall agreement was 79.54% and free marginal kappa was 0.74. CONCLUSION: There was substantial agreement among the observers as regards reproducibility of categories which can improve if we combine certain categories, especially "suspicious" and "malignant."

A report of two cases of dedifferentiated endometrioid carcinoma: A newly described underrecognized tumor of poor prognosis.

Dedifferentiated endometrioid carcinoma or dedifferentiated endometrioid adenocarcinoma (DEAC) is defined by the presence of undifferentiated carcinoma with endometrioid carcinoma. Undifferentiated component can be misinterpreted as solid component of high-grade endometrioid carcinoma or sarcomatous component of malignant mixed mullerian tumor. We present two cases of DEAC. Two postmenopausal women underwent hysterectomy for vaginal bleeding. Microscopically, sections from the endometrial tumors showed a biphasic growth consisting of an undifferentiated component and a glandular component with sharp transition between the two components. The undifferentiated component showed focal positivity for cytokeratin and vimentin, while glandular component was diffusely positive for cytokeratin and negative for vimentin expression.

The destructive spontaneous ingression of tunable silica nanosheets through cancer cell membranes.

Robust inorganic graphene analogues with atomic level sharp edges have seldom been investigated to decipher the interaction of two-dimensional materials with the cell membrane. Silica nanosheets (NSs) with four different thicknesses between 2.9 nm and 11.1 nm were synthesized by microwave irradiation and these colloidal NSs were able to spontaneously penetrate the cell membrane leaving membrane perforations at their sites of entry. The NS-ingression was most effective with the thinnest NSs, when studied in vitro. The atomistic details of the NS-membrane interaction were revealed by molecular dynamics (MD) simulations, which showed that the extraction of phospholipids was most favored when NSs were oriented vertically with respect to the membrane surface. While the folic acid modified NSs demonstrated a riveting tendency to penetrate the cancer cell membrane, co-modification with doxorubicin (DOX) unexpectedly reduced their capability. Migrating away from a conventional drug delivery approach, here we show that silica NSs can kill cancer cells primarily by mechanical scalpelling. Targeted ingress could be achieved through antibody conjugation on the NSs and thus only cancerous HeLa cells are affected by this treatment, leaving the normal HEK-293 cells intact. This destructive ingress through limited oxidative stress offers a previously unexplored route to treat fatal diseases without the necessity of transporting expensive drugs or radiation therapy, thereby bypassing deleterious side effects on healthy cells.

Cytology Microarray on Cell Block Preparation: A Novel Diagnostic Approach in Fluid Cytology.

BACKGROUND: The cytological examination of serous body effusions to diagnose and stage malignancy is well accepted in clinical medicine. Conventional smear (CS) and cell block (CB) study has to be complemented with immunohistochemistry (IHC) for a definitive diagnosis of malignancy and also to differentiate it from reactive mesothelial cells. Cytology microarray (CMA) is a modification of tissue microarray which involves core needle biopsy of multiple cell blocks and embedding it in a single block. AIM: The aim of this study was to assess the effectiveness of IHC technique in CMA for rapid diagnosis of malignancy and to reduce the cost of testing. MATERIALS AND METHODS: In this study, 82 pleural fluids were collected and subjected to CS and CB study followed by IHC in CMA blocks. Six commonly used antibodies were applied to confirm malignancy and diagnose the primary. RESULTS: Nineteen cases were diagnosed as malignancy by CB method. MOC-31 confirmed adenocarcinoma deposit in 67% cases of which 44% were proved to be of lung primary by TTF1. CONCLUSIONS: IHC on CMA blocks of effusion fluids is a very effective technique that can significantly reduce the cost of testing by >70%.

Galactocele of adult male breast: A cytopathologist's perspective.

Galactocele, although a common cytological diagnosis in females, is not previously reported as a cause of breast enlargement in adult males. Hyperprolactinemia is the principal cause of galactocele in male breast. Besides drug induced hyperprolactinemia, other anatomical lesions of hypothalamo-pituitary region and different medical conditions like cirrhosis and chronic kidney disease are to be considered along with a full evaluation of features revealing hypogonadism in case of galactocele. Aspirated milk from the male breast is the primary clue for this detailed investigation process. Here we are presenting the first case of galactocele of the male breast due to hypogonadotropic hypogonadism with idiopathic hyperprolactinemia.

Enhancement of anticancer activity and drug delivery of chitosan-curcumin nanoparticle via molecular docking and simulation analysis.

Computational analyses followed by traditional wet-bench experiments have become a method of choice due to successful results. To enhance the solubility and bioavailability of curcumin within chitosan nanoparticle, we have exploited computational methodologies i.e. docking, BBD-RSM and MD simulation for the polymer selection, NPs' formulation, optimization and their stability confirmation in an aqueous medium, respectively. Formulated CSCur NPs were assessed for in-vitro release, which exhibited a sustained release pattern and four-fold higher cytotoxic activity in a nanoparticulated system. Enhanced uptake, apoptotic effect of CSCur NPs were established by morphological changes in cells as observed by fluorescence microscopy and FE-SEM. DNA damage, cell-cycle blockage and elevated ROS levels further confirm the anticancer activity of the CSCur NPs following apoptotic pathways. In-vivo study on Danio rerio, for uptake and toxicity reveal the particle's biocompatibility and nontoxicity. Therefore, CSCur NPs could be the potential formulation for a safe chemotherapeutic drug for cancer.

Role of Cytology in Early Diagnosis of Cerebrotendinous Xanthomas.

Cerebrotendinous xanthomatosis is a rare autosomal recessive lipid storage disease characterized by widespread tissue deposition of two neutral sterols, cholestenol and cholesterol, resulting in tendinous xanthomas, juvenile cataracts, progressive neurological defects, and premature death from arteriosclerosis. Because it is a treatable cause of cerebellar ataxia and dementia, its early diagnosis is desirable. Here, we have reported the case of an 11-year-old boy with this disorder who was diagnosed based on the cytological findings of fine needle aspiration and clinical features.

Rhinosporidiosis of Parotid Duct Presenting as Consecutive Bilateral Facial Swelling: A Rare Case Report and Literature Review.

Rhinosporidiosis is a chronic granulomatous infection caused by Rhinosporidium seeberi. Sporadic cases of rhinosporidiosis has been reported from many countries but is endemic in Southern India (Madurai, Ramnad, Rajapalayam and Sivaganga), Nepal, Bangladesh and Sri Lanka. This disease commonly affects the mucous membrane of the nose or naso-pharynx and presents as a leafy, polypoidal mass. The reported extranasal sites include the oro-pharynx, eye, ear, larynx, trachea, bronchi, skin and genital mucosa. It may also become disseminated to present as a generalized form. In our case a 40-year-old female from rural West Bengal (Eastern India) presented with right sided facial swelling. Our provisional diagnosis was parotid duct cyst on the basis of careful history, scrupulous clinical examination and relevant investigations comprising CECT scan of face. Although Rhinosporidiosis was not taken into consideration in the clinical differential diagnosis, it was eventually diagnosed postoperatively by histopathological examination of surgical specimen. Two months later in follow-up, the same patient presented to us with left sided facial swelling. We managed the left facial swelling successfully with minimally invasive surgery and 100mg twice daily dapsone for 6 months. We present this case firstly because Rhinosporidiosis of parotid duct (stensen's duct) is a rare entity and secondly non-neoplastic cysts of the salivary glands are also uncommon and represent only 2-5% of all salivary gland lesions. Furthermore our case emphasizes that the clinicians should aware of this rare clinical entity specially in endemic areas, because minimally invasive techniques and medications might solve the problem while helping patients to avoid surgical excision.

Simultaneous knockdown of uPA and MMP9 can reduce breast cancer progression by increasing cell-cell adhesion and modulating EMT genes.

In cancer progression, proteolytic enzymes like serine proteases and metalloproteinases degrade the basement membrane enabling the tumor cells to invade the adjacent tissues. Thus, invasion and metastasis are augmented by these enzymes. Simultaneous silencing of uPA and MMP9 in breast cancer cells decreased the wound healing, migratory, invasive and adhesive capacity of the cells. After simultaneous down regulation, cells were seen to be arrested in the cell cycle. There was a remarkable increase in the expression of cell to cell adhesion molecule E-cadherin, and decrease in Vimentin and Snail expression. In addition, there was a significant decrease in the expression of the stem cell marker Oct-4. In the breast tumor samples it has been observed that, tumors, expressing higher level of uPA and MMP9, express less amount of E-cadherin. It has also been observed that few tumors also show, Vimentin positive in the ductal epithelial area. Thus, our model can help for checking the aggressive tumor invasion by blocking of uPA and MMP9. Our present observations also give the concept of the presence of aggressive epithelial cells with mesenchymal nature in the tumor micro-environment, altering the expression of EMT genes.

CT-guided aspiration cytology of advanced silicosis and confirmation of the deposited zeolite nano particles through X ray diffraction: A novel approach.

Silicosis is a common occupational lung disease, resulting in fibrotic nodular lesions in the upper lobes of the lung parenchyma. Most of the pneumoconioses are diagnosed on the basis of relevant history and clinico-radiological correlation. Image-guided aspiration cytology appears to be poorly yielding and is not usually considered as a diagnostic modality. However, silicosis may sometimes offer a diagnostic challenge because of its radiological resemblance and clinical overlap with pulmonary tuberculosis and neoplastic lesions. We present a unique situation where image-guided fine needle aspiration cytology (FNAC) has been advised on the basis of nodular upper lobe opacities. The cytology smears revealed hypocellular granular material, while phase contrast and polarized light microscopy highlighted crystalline particles. History of silica dust exposure long back was available after the cytological evaluation, suggesting the diagnosis of pulmonary silicosis. X ray diffraction (XRD) crystallography was also possible on cytology smears, confirming zeolite nano particles of size as small as 40 - 50 nm as the concerned agent for the first time. Cytological evaluation by phase contrast and polarized light microscopy may be useful for the confirmation of silicosis, supplemented by clinical history and radiological evaluation. XRD on smears may help in determination of chemical nature and particle size.

A study of the expression and localization of toll-like receptors 2 and 9 in different grades of cervical intraepithelial neoplasia and squamous cell carcinoma.

TLRs are important molecules of innate immune response, those play central role in host pathogen interaction and recognition through pathogen associated molecular patterns (PAMPs). Previous studies have indicated the role of TLRs in many human malignancy and cervical cancer in terms of viral recognition and inflammatory changes in-vivo. The objective of this study was to evaluate the expression and localization of toll-like receptor (TLR) 2 and TLR9 in preinvasive and invasive cervical cancer patients and to investigate its use as a probable diagnostic tool for better management cervical cancer. This single institution study includes individuals with normal, precancerous lesions, cervical intraepithelial neoplastic (CIN) and invasive squamous cell carcinoma (SCC) of the cervix. Upon confirmation by histopathology, fluorescence based immunohistochemistry was performed in all patients for TLR2 and TLR9, followed by semi-quantitative estimation of the staining intensity and grade of expression. The expression pattern of TLR2 and TLR9 does not vary greatly from normal to precancerous lesions, but a significant variation was observed in advance stages, i.e. squamous cell carcinoma of the uterine cervix. Additionally the expression increased marginally in higher grades. In spite of their low difference in expression along different stages of cervical cancer, both TLR2 and TLR9 could detect the disease at an advance stages as depicted by the receiver operator characteristics curve analysis.