Comparative validation of c-kit exon 11 mutation analysis on cytology samples and corresponding surgical resections of gastrointestinal stromal tumours

Objective:  Studies have shown that c-kit mutation analysis of gastrointestinal stromal tumours (GISTs) obtained by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) can be routinely performed. We validated c-kit exon 11 mutational analysis on cell block material obtained from fine needle aspiration cytology (FNAC) for diagnostic purposes and compared it with the same analysis in formalin-fixed paraffin-embedded full sections of the corresponding resection specimens.
Methods:  c-kit mutation analysis was done on cell block material obtained from ten cases encountered in our department from 1999 to 2008 on which FNAC was attempted pre-operatively. The findings were compared with analysis on full paraffin section of the corresponding resected tumours in seven cases where patients opted for resection. c-kit exon 11 was examined via bidirectional nucleic acid sequencing.
Results:  Our results showed 100% concordance for the presence and type of exon 11 mutation in the resected and aspirated tumours in all seven cases. These mutations had diagnostic value when compared with other neoplasms that are part of the cytomorphological differential diagnosis, such as leiomyosarcoma or gastric adenocarcinomas.
Conclusion:  Molecular cytopathology is a powerful tool that can complement morphology and immunohistochemical assessment of cytological material in routine practice for the diagnosis and prognostication of GISTs. We briefly discuss the advantages and limitations of the fine needle method of obtaining tissue for the diagnosis and prognostication of GISTs, and its current therapeutic strategies.     

Difficulties in diagnosing small round cell tumours of childhood from fine needle aspiration cytology samples

There are four basic reasons for the difficulties in diagnosing small round cell tumours (SRCT) in childhood from fine needle aspiration cytology (FNAC) samples. First, SRCTs are rare and it is difficult for cytopathologists to obtain enough experience for rendering reliable diagnoses. Second, SRCTs are morphologically very similar. Third, many SRCTs do not have specific antigens which could be demonstrated with immunocytochemistry (ICC) or they lose them when poorly differentiated. In addition, cross reactivity exists between some SRCTs. Unstandardized performance of ICC also contributes to the difficulties due to unreliable results. Fourth, suboptimal FNAC samples add additional pitfalls. The latter may be due to partly degenerate samples or to unrepresentative ones in cases where a SRCT is a heterologous component of another nosological entity. Lymphoma, neuroblastoma, nephroblastoma, Ewing's tumour/primitive neuroendocrine tumours and rhabdomyosarcoma are discussed in detail, while other less common SRCTs are mentioned as differential diagnoses when appropriate. The use of cytogenetic and molecular techniques for differentiating between certain SRCTs is helpful in some doubtful cases. However, there are still problems in the use of these techniques, especially their cost which may delay their being introduced in every cytopathology laboratory.     

Fine needle aspiration cytology of solitary fibrous tumours of the pleura

OBJECTIVE: To analyse fine needle aspirates from solitary fibrous tumour (SFT) of the pleura and to elucidate the cytological features unique to these tumours and differential diagnostic findings of benign and malignant SFTs. METHODS: Fine needle aspiration (FNA) cytology slides from eight cases of SFT of the pleura, including six benign and two malignant SFTs, were reviewed. The subsequent histological slides were also examined. RESULTS: Cytological diagnoses from six histologically proven cases of benign SFTs were low-grade sarcoma (one), non-small cell carcinoma (one), malignant tumour (1) and benign (three). Two cases of malignant SFTs were cytologically diagnosed as malignancy. The aspirates showed a varying degree of cellularity. Most smears were composed of single, scattered fusiform cells, and irregular loose aggregates of oval to spindle cells intimately admixed with dense collagenous stroma. Two malignant SFTs had a greater number of cells in clusters, and displayed mitotic activity, without significant cytological atypia. CONCLUSIONS: The diagnosis of SFT may be suggested by a combination of cytological and radiological findings. The precise determination of malignancy for SFT, however, is not usually straightforward on the basis of cytological features alone. The findings of highly cellular clusters and mitotic activity in the FNA cytological smear can help differentiate malignant from benign SFTs.     

Role of fine needle aspiration cytology in the diagnosis of soft tissue tumours and tumour-like lesions

In this study, we evaluated the usefulness of fine needle aspiration cytology (FNAC) in the diagnosis of soft tissue tumours. We have also assessed the various pitfalls of FNAC of soft tissue tumours. This was a retrospective study and here we analysed only 82 histopathology proven cases of FNAC of soft tissue tumours diagnosed in a five and half year period. On histopathological examination, 55 of these cases were malignant and 27 were benign. There was a total of 15 recurrences and histopathology was available prior to FNAC in only eight of these cases. Therefore, excluding these eight cases, malignant tumours were primarily diagnosed by FNAC in 47 cases. The sensitivity, specificity and positive predictive value of FNAC in diagnosis of soft tissue tumours were 91.5%, 92.5% and 95.5%, respectively. Only 22 of 47 cases (46.8%) were correctly categorized. There were two false-positive and four false-negative cases. One case each of fibromatosis and schwannoma were reported as sarcoma. False-negative cases were fibrosarcoma (1), malignant nerve sheath tumour (2) and haemangiopericytoma (1). FNAC was very useful in distinguishing benign from malignant soft tissue tumours. However, it was not so effective in exact categorization of tumours.     

Histoplasmosis: cytodiagnosis and review of literature with special emphasis on differential diagnosis on cytomorphology

N. Gupta, S. K. Arora, A. Rajwanshi, R. Nijhawan and R. Srinivasan
Histoplasmosis: cytodiagnosis and review of literature with special emphasis on differential diagnosis on cytomorphology Background: Human infection with Histoplasma capsulatum runs the gamut from asymptomatic to disseminated disease. In immunocompromised patients, a tiny inoculum can lead to widespread disseminated infection. Early diagnosis and initiation of treatment is therefore important. Objective: To review the cases of histoplasmosis diagnosed on fine needle aspiration cytology (FNAC) and to discuss the clinical presentation, associated inflammatory response, load of organisms and differential diagnosis on cytomorphology in these cases. Methods: Retrospective review of seven cases of histoplasmosis at a tertiary‐care centre during the period from 1998 to 2009 was performed. Clinical presentation along with cytomorphological features were studied and discussed in detail. Results: The mean age of patients was 48.6 years and six out of seven were male. History of immunodeficiency (HIV) was available in five cases. Six patients presented with peripheral and/or abdominal lymphadenopathy. One patient had nodular shadows in both lungs and two also had skin lesions. On cytological smears, a variable load of uniform round to oval, about 2–4 μm in diameter, budding yeasts were seen intracellularly (within histiocytes) as well as extracellularly. In one case (HIV positive), these organisms were also seen within neutrophil polymorphonuclear leucocytes. In two cases, an inflammatory response in the form of epithelioid cell granulomas along with multinucleated giant cells was seen. Conclusions: FNAC is a reliable tool to recognize infection with H. capsulatum in tissues. This infection can cause a variable inflammatory response, which should be considered while reporting on such cases.     

Analysis of proliferating cell fraction determined by monoclonal antibody to M1-subunit ribonucleotide reductase and Ki-67 in relation to p53 protein expression in fine-needle aspirates from non-Hodgkin's lymphomas

The purpose of this study was to analyse the proliferative fraction with the monoclonal antibody M1-R-R to M1-subunit ribonucleotide reductase and with MIB-1 to Ki-67 antigen in relation to p53 protein expression in fine needle aspirates from B-cell non-Hodgkin's lymphomas. One hundred and thirty-seven cases, previously diagnosed and sub-typed according to the Kiel classification and characterized by immunophenotyping, were included in the study. The M-1 subunit ribonucleotide reductase (M1-R-R), Ki-67 and p53 antigens were detected using monoclonal antibodies on stored cytospin preparations. There was a good correlation (r = 0.72) between Ki-67 and M1-R-R positive cell fraction in both high and low grade lymphomas. High-grade lymphomas had a median percentage of M1-R-R/MIB-1 positive cells of 53.0/73.0 for lymphoblastic, 61.0/52.0 for immunoblastic and 33.5/41.0 for centroblastic lymphomas, respectively. In low grade lymphomas figures of median percentage of M1-R-R/MIB-1 were 9.0/15.0 for centroblastic/centrocytic, 11.0/9.5 for chronic lymphocytic leukaemia, 16.0/27.0 for centrocytic and 12.0/9.0 for immunocytomas, respectively. The median percentages of M1-R-R/MIB-1 for high and low grade lymphomas were 37.0/50.5 and 11.0/12.0, respectively. In the p53 positive cases the proliferation rate as measured by staining for M1-R-R and MIB-1 was higher than in p53 negative cases, but the difference was not statistically significant. The results show that cytospin material obtained by fine needle aspiration and stored at -70 degrees C for years can be used reliably for both peroxidase-avidin-biotin and three-step alkaline phosphatase immunocytochemical staining. In addition, proliferation fraction determined by M1-R-R monoclonal antibody staining correlates well with that measured by an established marker for cell proliferation, the Ki-67 antibody. However, the proliferation fraction as measured by the two antibodies differs in the various subtypes of non-Hodgkin's lymphoma which indicates that they may contribute different prognostic information.     

Incidence and cytological features of pulmonary hamartomas indeterminate on CT scan

Objective:  Pulmonary hamartomas have a characteristic heterogeneous radiological appearance. However, when composed predominantly of undifferentiated mesenchymal fibromyxoid component, their homogeneous appearance on computed tomography is indeterminate for malignancy. Rendering an accurate preoperative diagnosis in these cases can alter management. The aim of this study was to determine the incidence and accuracy of cytodiagnosis for hamartomas 'indeterminate' by imaging.
Methods:  We retrospectively reviewed records for hamartomas diagnosed by transthoracic fine needle aspiration (FNA) including immediate impressions and final diagnoses. Cytological features evaluated included the presence of fibromyxoid stroma, bronchioloalveolar cell hyperplasia, fibroadipose tissue, cartilage and smooth muscle.
Results:  Eighteen (1.3%) hamartomas were identified from 1355 transthoracic FNAs. The immediate impression was hamartoma in 13 (72%), carcinoid in one (6%), mucinous bronchioloalveolar carcinoma in two (11%) and non-diagnostic in two (11%). The final diagnosis of hamartoma in cases diagnosed as carcinoid, mucinous bronchioloalaveolar carcinoma and non-diagnostic on immediate impression was rendered following assessment of all cytological material.
Conclusion:  Overall, FNAs are highly reliable for diagnosing hamartomas even when composed principally of undifferentiated mesenchymal fibromyxoid stroma, especially with the aid of all available preparations including Diff-Quik smears, Papanicolaou smears, ThinPreps and cell block material.     

Clear cell sarcoma, cellular mesoblastic nephroma and metanephric adenoma: cytological features and differential diagnosis with Wilms tumour

Wilms' Tumour (WT) is the most common kidney tumour in childhood, this fact and the embryonic complexity of WT create, whenever one of its three classical components predominates in cytologic smears, difficulties in the differential diagnoses with other less common entities. In the present study, we review the cytological and immunohistochemical characteristics of three children renal tumours, a Clear Cell Sarcoma of the Kidney (CCSK-case1), a Cellular Mesoblastic Nephroma (CMN-case2) and a Metanephric Adenoma (MA-case3) and compare them, for differential diagnostic purposes, with smears of blastematous, mesenchymal and epithelial predominant WTs, previously diagnosed in our Department. In all cases a mass was detected in the abdomen (2 and 8 year old children-cases 1 and 3, respectively), and pre-birth in case 2 (the tumour was detected during pregnancy). Fine needle biopsy was performed followed by routine cytologic examination. The presence of moderate amount of blue pale cytoplasm in neoplastic cells (case1), the presence of tightly cohesive, bland, spindle tumour cells (case2) and the identification of small, well differentiated epithelial tubules with psammoma bodies in case 3, were the main morphologic characteristics that we think represent the most important elements for distinguishing our cases from a WT. Immunoreactivity was only helpful in case 1 as we found a characteristic dot-like pattern positivity for vimentin, in the absence of immunoreactivity for the other markers that are usually positive in WT. Summing up, these three cases demonstrate that cytopathologists should be aware of the occurrence of uncommon renal neoplasms in childhood and should be acquainted with their characteristics, in order to avoid false diagnoses.     

Fine needle aspiration (FNA) in the diagnosis of soft tissue tumours; a review of 22 years experience

FNA plays an important role in preoperative diagnosis of soft tissue tumours. A close clinical/morphologic cooperation is essential. FNA should be performed on the most accessible part of the tumour, avoiding penetration of the deep portions of the tumour. Needles 0.7 mm (22 G) are recommended. For deep lesions, needles with a stylet should be used. After the FNA, tattooing of the aspiration channel is recommended, and the channel is surgically removed together with the tumour, if a sarcoma. Material from the FNA can be used for additional examinations, i.e. electron microscopy, immunohistochemistry, DNA ploidy analysis and chromosomal analysis. Those techniques are of great importance in the differential diagnosis, particularly in the paediatric small/round cell tumours. the majority of sarcomas can be defined as low grade or high grade malignant in FNA. For malignancy grading the following parameters are used: cellularity, pleomorphism, chromatin pattern, nucleolar structure, mitotic figures and necroses. Cytodiagnostic details of the most common soft tissue tumours and their differential diagnoses are presented.     

Diagnostic dilemmas of hyalinizing trabecular tumours on fine needle aspiration cytology: a study of seven cases with BRAF mutation analysis

T. Kim, Y. L. Oh, K. M. Kim and J. H. Shin Diagnostic dilemmas of hyalinizing trabecular tumours on fine needle aspiration cytology: a study of seven cases with BRAF mutation analysis Objective: Hyalinizing trabecular tumours (HTTs) are rare follicular‐derived neoplasms that behave in an almost benign manner. HTT is frequently misdiagnosed as papillary carcinoma by fine needle aspiration (FNA) cytology or as papillary or medullary carcinoma on surgical resection. Methods: The authors examined FNA material from seven cases of histologically verified HTT. Cytological findings were reviewed and correlated with ultrasonographic and histological features. In addition, MIB‐1 and calcitonin immunostaining was performed on surgical specimens, and BRAF mutation analysis on three pre‐operative FNA specimens and seven histology specimens. Results: The original cytological diagnosis was either suspicious or positive for papillary carcinoma in all patients. The FNA‐based differential diagnoses included HTT, papillary carcinoma or, less likely, medullary carcinoma in two patients. Aspirates showed oval to spindle‐shaped cells with frequent intranuclear inclusions, isolated in loosely cohesive groups with a trabecular or syncytial pattern in a bloody background. Radiating arrangements of tumour cells surrounding hyaline stroma with serrated calcifications and a lack of papillary or sheet‐like fragments may suggest HTT on FNA. Spherical calcified bodies and possible psammoma bodies were frequently found in three cases. Retrospectively, six of the seven cases showed membranous immunoreactivity for MIB‐1, but none of the seven possessed the BRAF (V600E) mutation or showed calcitonin reactivity. Conclusions: Although the recognition of HTT on FNA cytology is difficult, because of its morphological similarities to papillary and medullary carcinoma, its characteristic cytological features along with ultrasonographic findings may suggest the diagnosis preoperatively and avoid surgical over‐treatment.     

Expression of Ki-67, p53 and p63 proteins in keratocyst odontogenic tumours: an immunohistochemical study

Aim To investigate the immunohistochemical expression of Ki-67, p53 and p63 in Keratocyst Odontogenic Tumours (KOTs) in order to contribute to the biological profile of this tumor. Methods Immunohistochemical technique was performed using the EnVision™ System in 37 cases of KOTs. Results Ki-67- and p53-immunostained cells were mainly located in the suprabasal layers. p63-positive cells were found throughout the lining cystic epithelium. No difference in the immunostaining for these proteins was observed between primary and recurrent KOTs (Ki-67: P = 0.5591; p53: P = 0.9847; p63: P = 0.9127), or between KOTs associated with Nevoid Basal Cell Carcinoma Syndrome (NBCCS) and sporadic KOTs (Ki-67: P = 0.7013; p53: P = 0.3197; p63: P = 0.2427). Conclusions It is possible that biological behavior of KOTs may be related to suprabasal proliferative compartment in the cystic epithelium as observed by high levels of Ki-67, p53 and p63. In addition, p63 immunostaining may represent immaturity of keratinocytes in KOTs, and suggests that this protein may participate in the regulation of epithelial cell differentiation. Taken together, these data may favor tumorigenesis on KOTs.     

Bromodomain and extraterminal domain inhibitor enhances the antitumor effect of imatinib in gastrointestinal stromal tumours

In gastrointestinal stromal tumours (GISTs), the function of bromodomain‐containing 4 (BRD4) remains underexplored. BRD4 mRNA abundance was quantified in GISTs. In the current study, we investigated the role of BRD4 in GISTs. Our results show a significant enhancement in BRD4 mRNA and a shift from very low‐risk/low‐risk to high‐risk levels as per NCCN specifications. Overexpression of BRD4 correlated with unfavourable genotype, nongastric location, enhanced risk and decreased disease‐free survival, which were predicted independently. Knockout of BRD4 in vitro suppressed KIT expression, which led to inactivation of the KIT/PI3K/AKT/mTOR pathway, impeded migration and cell growth and made the resistant GIST cells sensitive to imatinib. The expression of KIT was repressed by a BRD4 inhibitor JQ1, which also induced myristoylated‐AKT‐suppressible caspases 3 and 9 activities, induced LC3‐II, exhibited dose‐dependent therapeutic synergy with imatinib and attenuated the activation of the PI3K/AKT/mTOR pathway. In comparison with their single therapy, the combination of JQ1/imatinib more efficiently suppressed the growth of xenografts and exhibited a reduction in KIT phosphorylation, a decrease in Ki‐67 and in the levels of phosphorylated PI3K/AKT/mTOR and enhanced TUNEL staining. Thus, we characterized the biological, prognostic and therapeutic implications of overexpressed BRD4 in GIST and observed that JQ1 suppresses KIT transactivation and nullifies the activation of PI3K/AKT/mTOR, providing a potential strategy for treating imatinib‐resistant GIST through dual blockade of KIT and BRD4.     

Role of fine needle aspiration cytology in the diagnosis and management of Warthin’s tumour of the salivary glands

J. M. Viguer, B. Vicandi, J. A. Jiménez‐Heffernan, P. López‐Ferrer, P. González‐Peramato and C. Castillo
Role of fine needle aspiration cytology in the diagnosis and management of Warthin’s tumour of the salivary glands Objective: Local excision surgical procedures and non‐surgical conservative management are considered alternatives to superficial parotidectomy in the treatment and management of Warthin’s tumour (WT). Such therapeutic alternatives demand accurate diagnosis. In order to determine whether fine needle aspiration cytology (FNAC) is capable of rendering such a minimally invasive diagnosis, we evaluated its accuracy and diagnostic parameters in a large series of histologically proven cases of WT. Methods: A cytohistological study of 116 salivary tumours from 110 patients (four WT were bilateral) with a histological or cytological diagnosis of WT. Results: Histology confirmed the cytological diagnosis in 103 of 114 tumours (90.4%). Two tumours were incorrectly diagnosed on cytology as WT. In 11 cases of WT there was an erroneous or non‐representative cytological diagnosis. The sensitivity was 90.4%, and positive predictive value 98.1%. Regarding malignancy, there were three misdiagnoses. One tumour diagnosed as WT was a low‐grade mucoepidermoid carcinoma. Two cases considered ‘suspicious of squamous cell carcinoma’ corresponded to WT. After review, 81.3% of the cases of WT were considered typical and 18.7% non‐typical; all misdiagnoses were in the latter group. Cytological difficulties could be divided into three areas: (i) absence of one or more diagnostic components; (ii) ‘squamoid’ pattern; and (iii) mucinous metaplasia. Degenerated oncocytes were present in 65% of cases. Conclusions: FNAC offers the possibility of a reliable diagnosis of WT. Pathologists must pay attention to the squamous appearance of degenerated oncocytes. Cytology, when coupled with clinical and image findings, may permit conservative tumour management.