Microarray based gene expression profiling of advanced gall bladder cancer.

BACKGROUND: Gall bladder cancer (GBC) is an aggressive cancer with specific predilection like female gender and specific geographical areas, however the molecular mechanisms and factors contributing to the clinical or biological behavior are not understood. AIM: The aim of this study was to perform a comprehensive analysis of differentially expressed genes in advanced GBC and chronic cholecystitis (CC) cases. MATERIALS AND METHODS: Microarray was planned on fresh specimens of advanced GBC and CC cases using single color cRNA based microarray technique (8X60K format; Agilent Technologies, USA). Twelve advanced GBC and four CC patients were included in the study. RESULTS: Of the total of 1307 differentially expressed genes, 535 genes were significantly upregulated, while 772 genes were significantly downregulated in advanced GBC vs CC samples. Differentially expressed genes were associated with biological processes (55.03%), cellular components (31.48%), and molecular functions (13.49%) respectively. The important pathways or key processes affected were cell cycle, DNA replication, oxidative stress, gastric cancer pathway. Using in silico analysis tools, three differentially expressed genes i.e. TPX2, Cdc45 and MCM4 were selected (for their significant role in DNA replication and microtubule function) and were further validated in 20 advanced GBC cohort by immunohistochemistry. Significant positive association of Cdc45 and MCM4 proteins was found in advanced GBC cases (p = 0.043), suggesting the probable oncogenic role of Cdc45 and MCM4 proteins in advanced GBC. CONCLUSION: Our data demonstrate the potential regulation of Cdc45-MCM4 axis in advanced GBC tumors. Additionally, our study also revealed a range of differentially expressed genes (e.g. TPX2, AKURA etc.) between GBC and CC, and further validation of these genes might provide a potential diagnostic or therapeutic target in future.

Mucinous adenocarcinoma of gallbladder: Subcategorisation on fine-needle aspiration cytology.

BACKGROUND: Mucinous adenocarcinoma (MC) of gallbladder is a rare histological subtype of gallbladder carcinoma (CaGB) which presents at an advanced stage and is associated with a poor prognosis compared to the conventional CaGB. This variant has been described mostly as reports or series, except for a single detailed histological and immunohistochemical analysis. Till date, there are no studies describing the cytomorphology of MC in detail. Hence, we undertook this study to analyse the cytomorphological features of MC. METHODS: A retrospective cytomorphological analysis was performed on MC identified out of all CaGB diagnosed on cytology over a period of last 4 years. The architectural and cellular features were recorded in a structured proforma. RESULTS: Thirty-three cases (33/987, 3.3%) were identified as MC. Extracellular mucin >90% was seen only in 3 cases whereas the remaining 30 had 50%-90% mucin. The predominant architectural pattern was tight epithelial fragments (14/33). The tumour cells were mostly of intermediate size (31/33) and had moderate amount of cytoplasm (31/33). Majority of the cases showed moderate nuclear pleomorphism (28/33) and nuclear chromatin was fine granular (17/33) or vesicular (14/33). Most of the cases had single and small nucleoli (26/33). Presence of inflammation composed predominantly of polymorphs was noted in 25 cases. Majority of the cases showed no (15/33) or scant necrosis (13/33). CONCLUSION: The morphological features of MC can very well be demonstrated on cytology. As they are associated with poor prognosis compared to conventional CaGB, cytopathologists should try to document the subtype.

Cytopathology of non-invasive follicular thyroid neoplasm with papillary-like nuclear features: A comparative study with similar patterned papillary thyroid carcinoma variants.

OBJECTIVE: Noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) is a recently described, indolent thyroid tumor, with well-defined histopathological diagnostic criteria. Cytology features are not well documented. We reviewed cytology of histologically proven cases of NIFTP and some of its common differentials to look for salient diagnostic features. METHODS: Cases reported on histopathology as follicular variant of papillary thyroid carcinoma (FVPTC), or NIFTP between July 2015 and April 2017 having available cytology smears were retrieved and reclassified as NIFTP, FVPTC, and classical papillary thyroid carcinoma with predominant follicular pattern (PTC-FP). Cytological features were assessed, classified as per The Bethesda System for Reporting Cytopathology and compared. RESULTS: There were 23 NIFTP cases, 18 FVPTC and 8 PTC-FP. A microfollicle-predominant pattern was seen in all. Nuclear score was 2 in most NIFTP cases (61%). Pseudoinclusions were absent. NIFTP showed features of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) (III) in 61%, follicular neoplasm/suspicious for a follicular neoplasm (FN/SFN) (IV) in 35% and suspicious for malignancy (SFM) (V) in 4%. Most of the FVPTCs were also called FN/SFN (IV) (56%) or AUS/FLUS (III) (22%). Nuclear features did not statistically differ from NIFTP. PTC-FP showed high-grade cytology in 75%, and higher nuclear score (3 in 75%) in contrast to NIFTP (P = .003). CONCLUSION: NIFTP and FVPTC show a similar distribution among the Bethesda categories hence precluding conclusive distinction on cytology. PTC-FP, in contrast, was found to have a statistically significant higher nuclear score and more commonly showed malignant cytology.

Adenocarcinoma predominant pattern subtyping and nuclear grading in cytology: Is there a role in prognostication of advanced pulmonary adenocarcinomas?

INTRODUCTION: Primary lung adenocarcinomas (ADs) show varied architectural patterns, and pattern-based subtyping of ADs is currently recommended due to prognostic implications. Predicting AD patterns on cytology is challenging; however, cytological nuclear features appear to correlate with histological grade and survival in early stage lung ADs. The feasibility and value of AD pattern prediction and nuclear grading on cytology in advanced lung ADs is not known. We aimed to predict patterns and analyse nuclear features on cytology and evaluate their role in prognostication. METHODS: One-hundred patients of Stage III/IV lung AD with available matched cytology and histology samples were included. Cyto-patterns based on cell arrangement patterns (flat sheets vs three-dimensional clusters vs papillae) and cyto-nuclear score based on nuclear features (size, shape, contour), nucleoli (macronucleoli vs prominent vs inconspicuous), and nuclear chromatin were determined, and correlated with predominant histological-pattern observed on the matched small biopsy and outcome. RESULTS: Higher cyto-nuclear scores were observed with high-grade histo-patterns (solid, micropapillary and cribriform), while the predicted cyto-patterns did not correspond to the predominant pattern on histology in 77% cases. Highest cyto-histo agreement was observed for solid pattern (72%). High grade histo-patterns and cyto-nuclear scores > 3 showed a trend towards inferior survival (not significant). CONCLUSIONS: Nuclear grade scoring on cytology is simple to perform, and is predictive of high grade patterns. Its inclusion in routine reporting of cytology samples of lung ADs may be valuable.

An institutional experience with The Paris System: A paradigm shift from ambiguous terminology to more objective criteria for reporting urine cytology.

BACKGROUND: Urine cytology is a highly specific modality for diagnosing high-grade urothelial carcinoma (HGUC), but plagued by low sensitivity and wide inter-observer variability mainly ascribed to the lack of an established template of reporting. The Paris System (TPS) working group proposed such a template at the 2013 International Congress of Cytology, replete with objective criteria for categorising specimens into one of the seven categories: non-diagnostic, negative for HGUC, atypical urothelial cells, suspicious for HGUC, HGUC, low-grade urothelial neoplasm and others (including non-malignant entities). This study was undertaken to determine the impact of TPS criteria in the morphological interpretation of urine samples. METHODS: A total of 255 voided urine specimens from 97 patients who had follow-up biopsies were re-assessed according to TPS criteria and correlated with the final histological diagnosis. RESULTS: Sixty-three patients were diagnosed with HGUC, and 34 had low-grade papillary UC on biopsy. Earlier samples from 40 (41.2%) patients were categorised as merely "atypical" wheereas the "positive for malignancy" category was assigned to 33 (34%) patients. After re-evaluation of the same cases using TPS criteria, cytological features in 44 (69.8%) out of 63 HGUC patients were correctly recognised as HGUC and samples from additional seven patients were re-categorised as suspicious for HGUC (total 81%). The sensitivity of the HGUC category in predicting HGUC was 69.8% which rose to 81% when HGUC was grouped with suspicious for HGUC category. CONCLUSION: The criteria outlined by TPS facilitated the standardisation of urine cytology reporting and significantly increased the sensitivity of diagnosing HGUC.

Copy number variations in testicular maturation arrest.

Testicular maturation arrest is characterized by interruption of germ cell development and differentiation. Genetic factors play important role in the causation of human disease, including male infertility. The objective was to study copy number variations in testicular maturation arrest using single nucleotide polymorphism (SNP) microarray technique. Conventional cytogenetics, targeted fluorescence in situ hybridization (FISH) and sequence-tagged site (STS) polymerase chain reaction (PCR) were used to confirm some of the SNP microarray findings. SNP microarray on 68 cases of testicular maturation arrest detected copy number variations (CNVs) mostly on sex chromosomes involving pseudoautosomal regions (PAR) 1, 2 and 3 as well as azoospermic factors (AZFs) besides three cases of chromosomal abnormalities (two Klinefelter syndromes and one case of dicentric Y). The AZF deletion was observed in 14 (20.6%) cases and the AZFc gain was observed in 6 (8.8%) cases. PAR 1 and 2 CNVs was observed in 5 (7.3%) cases. PAR 3 CNVs was detected in 19 cases and 2 controls. The TSPY2 gene gain (within PAR 3 CNVs) was observed in 16 cases and 1 control. CNV containing autosomal genes possibly associated with male infertility in this study was SPATA31A2-A5 (9p12) in five cases. In this study, SNP microarray identified possible underlying aetiology in 55.9% (38/68) cases besides identifying minimal critical region of AZFc deletion as 0.51 mb (Y:24356128-24873665) involving TTY5, RBMY2FP, RBMY1F, RBMY1J, TTY6 and PRY genes. SNP microarray seems superior, sensitive, specific as well as cost-effective method and has potential to be the first tier investigations to explore underlying genomic factors of testicular maturation arrest. The present study is an attempt to find out probable genomic factors with idiopathic testicular maturation arrest.

Fine needle aspiration cytology features of poorly differentiated thyroid carcinoma.

BACKGROUND: Poorly differentiated thyroid carcinoma (PDTC) is an uncommon thyroid malignancy with biological behaviour intermediate between well-differentiated and undifferentiated thyroid carcinoma. The cytological diagnosis of PDTC is often difficult as a result of a lack of well-established cytomorphological features and a considerable degree of morphological overlap with other commoner thyroid neoplasms. OBJECTIVE: To review the cytomorphological features of PDTC with the aim of highlighting salient diagnostic morphological features and differential diagnostic problems. METHODS: Seven cases of histologically proven PDTC with available aspiration cytology smears were reviewed for the presence of distinguishing cytomorphological features. RESULTS: The architectural arrangement of tumour cells was the most important diagnostic parameter. Cellular nests, three-dimensional clusters dyscohesive aggregates and singly dispersed cells in the background were present in all cases. A unique 'garlanded appearance', owing to the peripheral orientation of nuclei within the tumour cell clusters, was noted (71.4%) along with the presence of basement membrane-like material (71.4%). Transgressing vessels (85.7%) and endothelial wrapping of cell clusters (71.4%) were also noted. Interestingly, most cases lacked necrosis and mitotic activity that are included in the histological diagnostic criteria for PDTC. CONCLUSION: Although PDTC has considerable cytomorphological overlap with well-differentiated thyroid tumours, this present study highlights certain cytomorphological features that may suggest the correct pre-operative diagnosis, important for the appropriate management.

Cytomorphology of neuroendocrine tumours of the gallbladder.

OBJECTIVE: Gallbladder neuroendocrine tumours (GB-NETs) are rare and account for 0.5% of all NETs. GB-NETs have an aggressive behaviour, which depends on the tumour grade. The cytomorphological spectrum of these tumours has never been described in detail. The present study evaluates the cytological features of GB-NETs and grades them according to the World Health Organization (WHO) classification. Furthermore, the expression of thyroid transcription factor-1 (TTF-1) has not been investigated previously in GB-NETs, although found in a subset of extrapulmonary NETs. METHODS: Twenty cases of GB-NET among 875 gallbladder carcinomas diagnosed by ultrasound-guided fine needle aspiration cytology (FNAC) over a period of nearly 4 years were studied. The following parameters were evaluated: architectural pattern, nuclear chromatin, nucleoli, mitoses, necrosis, moulding, apoptosis and smudge cells. Cases were categorized into well-differentiated (grades 1 and 2), small cell carcinoma (SCC) (grade 3) and mixed adenoneuroendocrine carcinoma. Nuclear positivity for TTF-1 was considered as positive. RESULTS: Morphologically, tumour cells were mainly arranged in rosettes in the well-differentiated category; sudden anisonucleosis and rare nuclear moulding with or without mitotic figures were other features. Eleven cases of SCC showed prominent nuclear moulding with frequent smudge cells, mitoses, apoptosis and necrosis. Three mixed adenoneuroendocrine carcinomas showed papillary fragments and an acinar arrangement of tumour cells. Four of the nine SCCs in which TTF-1 was evaluated on de-stained smears showed nuclear positivity. Histopathology was available in two SCCs and showed morphology similar to FNAC. CONCLUSION: Cytology plays an important role in the diagnosis of GB-NETs for appropriate subtype characterization, which is necessary for the prognostication of these tumours. TTF-1 may not be used for the differentiation of gallbladder SCCs from pulmonary SCCs.

Differentiation of histoplasma and cryptococcus in cytology smears: a diagnostic dilemma in severely necrotic cases.

OBJECTIVE: The correct identification of fungal organisms is important for the appropriate clinical management of patients. It becomes difficult in necrotic smears when the tissue response is not clearly discernible. It is difficult to distinguish between histoplasma and cryptococcus in severely necrotic cases, where both appear as variably sized clear refractile haloes. METHODS: Four cases of adrenal necrotic histoplasma infection were studied and the morphology was compared with that of non-necrotic histoplasmosis and cases of cryptococcal infection. Eleven cases were analysed in fine needle aspiration cytology (FNAC) smears. Ziehl-Neelsen (ZN) stain was performed to exclude tuberculosis in necrotic smears. A clinical and serology correlation was performed where available. RESULTS: Necrotic cases of histoplasma infection revealed negative refractile clear haloes similar to those of cryptococcus. Histoplasma showed methylene blue-stained organisms in ZN stains, whereas the cryptococcus cases were negative. Similar methylene blue-stained organisms were seen in non-necrotic histoplasma infection. CONCLUSION: As a result of morphological overlap between cryptococcus and histoplasma, the distinction between the two fungi can be difficult in many cases. ZN staining appears to have a role in the differentiation of these fungi in severely necrotic cases. This observation needs to be validated on a larger number of cases with complete correlation with clinical, serology and treatment records.

Management and outcomes in massive bilateral Wilms' tumors.

PURPOSE: To evaluate the outcome of children with bilateral Wilms' tumor (BWT) treated on All India Institute of Medical Sciences-Wilms Tumor-99 (AIIMS-WT-99) protocol. MATERIALS AND METHODS: All children with BWT, registered in our solid tumor clinic from August 1999 through December 2010 were included. RESULTS: Of the 178 fresh cases of Wilms Tumor (WT) treated during this period, 11 (6.2%) had bilateral involvement. All patients except one (12 and 3 cm), had massive bilateral tumors of more than 10 cm on each side. There were eight boys and three girls in the age range 6-30 months. One patient had Denys-Drash syndrome. Twenty renal units were operated upon (12 tumorectomy, five partial nephrectomy, and three nephrectomies), while one patient with inferior vena cava (IVC) thrombus died of renal failure. Tumor spill occurred in three units, lymphnode was positive in two patients. Local recurrence occurred in four patients (six of 18 renal units (33%)-two bilateral and two unilateral). There was one recurrence in the liver that was treated with radio-frequency ablation. The 5-year overall survival (OS) was 90% (95% confidence interval (CI) = 50.8-98.6) and the relapse free survival (RFS) was 38% (95% CI = 6.1-71.6). CONCLUSION: Massive BWT respond poorly to preoperative chemotherapy, are often not amenable to partial nephrectomy/tumorectomy and have a higher local recurrence rate, giving a poor RFS.

Cell blocks in cytopathology: a review of preparative methods, utility in diagnosis and role in ancillary studies.

The cell block (CB) is a routine procedure in cytopathology that has gained importance because of its pivotal role in diagnosis and ancillary studies. There is no precise review in the published literature that deals with the various methods of preparation of CB, its utility in diagnosis, immunocytochemistry (ICC) or molecular testing, and its drawbacks. An extensive literature search on CB in cytology using internet search engines was performed for this review employing the following keywords: cell block, cytoblock, cytology, cytopathology, methods, preparation, fixatives, diagnostic yield, ancillary and molecular studies. Ever since its introduction more than a century ago, the CB technique has undergone numerous modifications to improve the quality of the procedure; however, the overall principle remains the same in each method. CBs can be prepared from virtually all varieties of cytological samples. In today's era of personalized medicine, cytological specimens, including CBs, augment the utility of cytological samples in analysing the molecular alterations as effectively as surgical biopsies or resection specimens. With the availability of molecular targeted therapy for many cancers, a large number of recent studies have used cytological material or CBs for molecular characterization. The various techniques of CB preparation with different fixatives, their advantages and limitations, and issues of diagnostic yield are discussed in this review.

Utility and pattern of positivity of p40 in the diagnosis of squamous cell carcinoma of the lung by cytology: the first study on fine needle aspiration smears.

OBJECTIVE: Immunohistochemistry for p40 has emerged as a clinically applicable tool with high sensitivity and specificity to distinguish lung adenocarcinoma and squamous cell carcinoma (SCC). It appears to be an excellent marker for squamous differentiation. Although application of p40 in cell blocks has been reported, its expression has not been described in cytology smears. The aim was to study the expression of p40 in fine needle aspirates of SCC of the lung and to analyse differences in immunoreactivity in variably differentiated SCC. METHODS: The study used aspirates of lung masses diagnosed as SCC over a period of 2 years. p40 immunocytochemistry was performed on destained Papanicolaou smears. Nuclear staining was semi-quantitatively evaluated as 0, 1 + , 2 +  and 3 +  based on the percentage positivity of tumour cells and was correlated with differentiation of the tumour. Adequate unmatched histology (50 biopsies) and cytology (25 smears) controls were taken for comparison. RESULTS: A total of 45 cases of primary and five cases of metastatic pulmonary SCC were identified. There were 17 well, 24 moderately and nine poorly differentiated SCC. p40 immunoreactivity was 2-3 +  in all moderate and poorly differentiated tumours, however, negative to 1 +  in all well-differentiated carcinomas (P < 0.0001). CONCLUSION: p40 immunostain is a valuable stain in identifying lung SCCs and works well in aspiration smears. The pattern of positivity varies with the differentiation of the tumour and is seen prominently in higher grade SCC where in practice the need arises for distinguishing them from either poorly differentiated adenocarcinomas or non-small cell carcinoma, not otherwise specified.

Cytopathology of neoplastic meningitis: A series of 66 cases from a tertiary care center.

BACKGROUND: Neoplastic meningitis (NM) is a condition characterized by leptomeningeal involvement by metastatic carcinoma. Detection of exfoliated malignant cells in cerebrospinal fluid (CSF) due to meningeal metastasis is frequently associated with diverse neurologic presentations. MATERIALS AND METHODS: In this retrospective study of all cases of NM diagnosed in CSF samples over a 20-year period at a tertiary care referral center, the cytomorphologic features were reviewed. RESULTS: Sixty six cases of NM were identified of which 36 already had an established diagnosis of malignancy while in 30 patients, there was no previously known tumor. The most common known primary in the former group was breast followed by ovary. Single cell pattern, cellular cannibalism, moderate cytoplasm and rounded nuclei were seen in breast and lung tumors. Papillary architecture and cytoplasmic vacuolation were seen in the ovarian primaries. Melanin pigment was seen in malignant melanoma. CONCLUSION: CSF cytology is an important tool for diagnosis of NM. Cytomorphologic features helped in diagnosis and for prediction of the primary site. Correct identification of this condition is important as it has therapeutic and prognostic implications.