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– Malignant mesothelioma (MM) is a rare aggressive neoplasm arising from mesothelial lining of body cavities, most commonly pleura and peritoneum. It is characterised by a poor prognosis and limited treatment options. A universally recognised risk factor for the development of MM is exposure to asbestos. However, evidence supporting a genetic susceptibility to the development of MM has been accumulating during the last decades. Intensive research for the identification of MM susceptibility genes has led to the discovery of BAP1 and to the definition of the so-called “BAP1-related tumour predisposition syndrome”. Patients carrying germline BAP1 mutations have an increased risk for the early development of tumours, including MMs, uveal melanomas, cutaneous melanocytic lesions, clear cell renal cell carcinomas and basal cell carcinomas. Furthermore, pathogenic variants in tumour suppressor genes with a role in DNA repair have been recently described in families with clustered MM cases. These genetic alterations seem to confer exaggerate sensitivity to asbestos carcinogenic effect and, arguably, increased response to specific chemotherapeutic strategies. While the translational significance of BAP1 alterations is explored in the research field, the identification of families carrying germline BAP1 mutations is mandatory to start appropriate surveillance programs and guarantee the best clinical management to these patients.

AimsSeveral predictive biomarkers of response to specific inhibitors have become mandatory for the therapeutic choice in non-small-cell lung cancer (NSCLC). In most lung cancer patients, the biological materials available to morphological and molecular diagnosis are exclusively cytological samples and minimum tumour wastage is necessary. Multiplex fluorescence in situ hybridisation (mFISH) to detect simultaneously ALK-rearrangement and ROS1-rearrangement on a single slide could be useful in clinical practice to save cytological samples for further molecular analysis. In this study, we aim to validate diagnostic performance of multiplex ALK/ROS1 fluorescence in situ hybridisation (FISH) approach in lung adenocarcinoma cytological series compared with classic single break apart probes.MethodsWe collected a series of 61 lung adenocarcinoma cytological specimens enriched in tumours harbouring ALK-rearrangement and ROS1-rearrangement. ALK and ROS1 status were previously assessed by classic FISH test using single break apart probes and immunohistochemistry. Study population was composed of 6 ALK-positive, 2 ROS1-positive and 53 ALK/ROS1-wild type. All specimens were analysed by multiplex FISH assay using FlexISH ALK/ROS1 DistinguISH Probe Zytovision.ResultsThe dual ALK/ROS1 FISH probe test results were fully concordant with the results of previous single ALK and ROS1 FISH tests on two different slides. 6 ALK-positive and 2 ROS1-positive were confirmed through multiplex FISH test, without false-positive and false-negative results. Multiplex ALK/ROS1 FISH test results agreed with immunohistochemistry assay staining results.ConclusionMultiplex ALK/ROS1 FISH probe test is a useful tool to detect simultaneously ALK-rearrangement and ROS1-rearrangement on a single slide in cytological specimens with a small amount of biomaterial.

Background: Renal cell carcinoma (RCC) constitutes 3% of all cancers, with a higher incidence in patients with age between 60 and 70 years. RCC frequently present as a metastatic tumor at diagnosis, and bones represent one of the most frequent sites. Many cases, mainly in young patients, includes the Xp11 translocation RCC. The cytological diagnosis of Xp11 translocation RCC in adult population it is rarely performed, likely for the morphological overlap with other adult renal cell carcinoma subtypes. Methods: We retrospectively analyze a series of 92 adult patients with metastatic bone tumors, diagnosed on fine-needle aspiration cytology (FNAC) samples, focusing mainly on the cytological, immunophenotypic and molecular features of Xp11 translocation RCC. Results: In our series 6 of 92 (6.5%) cases were metastatic RCC (mRCC), among them 2 cases were metastasis from Xp11translocation RCC. Those cases showed a bloody background, with several groups of atypical cells arranged in syncytial groups or in papillary groups composed by atypical cells with abundant cytoplasm, with scattered clear cells. TFE3 was positive on immunocytochemical analysis and specific translocation t(Xp11.23) was detected by FISH analysis. Conclusions: In adult patients with mRCC, it is necessary to consider also Xp11 translocation RCC among the diagnostic hypotheses. FNAC represents a valid tool to investigate bone lesions but cytological features of Xp11 translocation RCC are still poorly described and must necessarily be better defined.

OBJECTIVE: Anaplastic lymphoma kinase (ALK) gene has been demonstrated to be rearranged, mutated or amplified in several haematological and solid tumors. Moreover, the use of ALK inhibitors has recently revolutionized the treatment of ALK-rearranged patients affected by non-small cell lung carcinoma. Herein we review the genetic alterations of ALK in melanocytic neoplasms described in literature, focusing on their potential diagnostic and predictive role. MATERIALS AND METHODS: The Authors reviewed the pertinent literature through research on PubMed server was performed typing the terms "ALK", "Anaplastic lymphoma kinase", "ALKATI", "Melanoma", "Spitz", "Spitzoid". RESULTS: ALK translocations were demonstrated in melanocytic neoplasms, particularly in acral melanoma and spitzoid tumors. ALKATI was described in primary and metastatic melanoma, indicating its early occurrence in oncogenesis, with varying immunohistochemical expression of the protein. CONCLUSIONS: The identification of the specific type of ALK mutations could be interesting for planning biologic therapy of melanoma patients. Further studies are needed to evaluate the possibility to introduce an ALK-targeted therapy in patients affected by malignant melanoma.

Soft tissue tumors comprise a great variety of common and rare entities with overlapping features. Their diagnosis is based on the evaluation of several histological parameters which are difficult to assess on small incisional biopsies. Useful diagnostic markers in the field of soft tissue tumors include: 1) molecular biomarkers detecting pathogenetically relevant, distinctive alterations; 2) immunohistochemical surrogate biomarkers of pathogenetically relevant, distinctive molecular alterations; 3) highly specific immunohistochemical biomarkers indicating tumor differentiation. Their introduction in clinical practice has revolutionized the pre-operative diagnosis of soft tissue tumors. Cytology has long been considered inadequate as a first-line approach in this setting. However, since the implementation of new immunohistochemical and molecular tests with high diagnostic specificity, fine needle aspiration cytology (FNAC) is starting to gain acceptance for the pre-operative assessment of soft tissue tumors. FNAC represents a versatile, poorly expensive and well-tolerated diagnostic strategy with relevant advantages over histological biopsies. Moreover, evidences suggest that, in expert hands, FNAC can also aim at a definite diagnosis, especially if a cell block is prepared, allowing the application of multiple ancillary techniques.

The identification of biomarkers on cancer tissue samples could be obtained through several technologies. In this setting, the immunohistochemistry and in situ hybridization are accessible in most pathology laboratories. Particularly, immunohistochemistry can be used not only for diagnostic issues, but also to define prognostic classes and to define response to specific therapies. Particularly the last applications have been firstly developed in the breast cancer pathology. In addition, the development of molecular classification proposed some prognostic/predictive classes that could be easily defined by immunohistochemistry. Thus, the role of the pathologists has become increasingly important in the definition of prognosis and in the choice therapy, because the immunohistochemical biomarkers are used to guide treatment, to classify breast cancer into biologically and prognostically distinct subtypes. In this review, we will provide information on the current application of the immunohistochemical biomarkers useful in the management of breast cancer patients. Moreover, we consider the application of immunohistochemistry in the definition of the most promising biomarkers derived from molecular studies of the breast cancer, that in the future could integrate the characterization of breast cancer into clinical practice.

In the era of precision medicine, the identification of several predictive biomarkers and the development of innovative therapies have dramatically increased the request of tests to identify specific targets on cytological or histological samples, revolutionizing the management of the tumoral biomaterials. The Food and Drug Administration (FDA) has recently approved a selective neurotrophic tyrosine receptor kinase (NTRK) inhibitor, larotrectinib. Contemporarily, the development of multi-kinase inhibitors with activity in tumors carrying TRK fusions is ongoing. Chromosomal translocations involving the NTRK1, NTRK2, and NTRK3 genes result in constitutive activation and aberrant expression of TRK kinases in numerous cancer types. In this context, the identification of tumors harboring TRK fusions is crucial. Several methods of detection are currently available. We revise the advantages and disadvantages of different techniques used for identifying TRK alterations, including immunohistochemistry, fluorescence in situ hybridization, reverse transcriptase polymerase chain reaction, and next generation sequencing-based approaches. Finally, we propose a diagnostic algorithm based on histology and the relative frequency of TRK fusions in each specific tumor, considering also the economic feasibility in the clinical practice.

Angiosarcomas are ubiquitous neoplasms involving both cutaneous and soft tissue and visceral locations. Accumulating biomolecular evidences suggest that cutaneous angiosarcomas are distinctive entities with molecular, clinical and pathological peculiarities. Despite several ongoing clinical trials with promising therapeutic agents, the prognosis of cutaneous angiosarcomas is dismal and survival still rely on early diagnosis and surgery. An accurate diagnosis and the knowledge of the underlying molecular landscape are therefore essential to improve the prognosis. We detail the molecular, clinical, dermoscopic, morphological and prognostic features of cutaneous angiosarcoma. Although the molecular landscape of cutaneous angiosarcoma is not completely understood, accumulating evidences suggest that there are characteristic molecular alterations including dysregulation of angiogenesis and several complex molecular pathways. Secondary cutaneous angiosarcomas, arising in correlation with chronic lymphedema and ionizing radiation, have different molecular hallmarks, which are also leading to the first diagnostic applications. The diagnosis of cutaneous angiosarcoma may be challenging, as well-differentiated forms can be hard to distinguish from benign and low-grade vascular neoplasms, while poorly differentiated forms can be easily confounded with other non-vascular high-grade neoplasms. An accurate and early diagnosis, which is mandatory to ensure the best survival for the patients, is mainly based on morphological hallmarks.

Myoepithelioma represents less than 1% of all salivary glands tumors. Generally are benign tumors localized in parotid gland. Only FNAC, often, is insufficient for the correct diagnosis of the lesion, which can be confused with a pleomorfic adenoma or a malignant myoepithelial carcinoma. Because of its rarity, we describe a case report of parotid gland myoepithelioma treated, successfully, with total parotidectomy and preservation of the facial nerve.

Coffee is one of the most widely drunk beverages in the world. In recent years the consumption of instant coffee has increased faster when compared to roasted coffee and today plays an important role in the world economy. The traditional production process of soluble coffee involves several stages, including percolation at temperatures above 180cCand stabilization by means of spray-drying or lyophilization. These high temperatures may be responsible for the degradation of the molecules extracted, in particular the aromatic ones. For these reasons, the aim of this work was to compare, from an analytical and sensorial point of view, coffee made from the Arabica variety prepared using the traditional method compared with others obtained by applying high pressure and water at room temperature. The results showed that the best extraction yields, in terms of soluble solids (g/100g) and total polyphenol (mg gallic acid/100 g) were found in samples of arabica coffee treated with hot water. However the coffee obtained at a pressure equal to 8 bar for 2 h was judged as the most valid from a sensory point of view.

BACKGROUND: Lymph nodal involvement is an important clinical-pathological sign in primary cutaneous lymphoma (PCL), as it marks the transformation/evolution of the disease from localized to systemic; therefore the surveillance of lymph nodes is important in the staging and follow up of PCL. Fine needle cytology (FNC) is widely used in the diagnosis of lymphadenopathies but has rarely been reported in PCL staging and follow-up. In this study an experience on reactive and neoplastic lymphadenopathies arisen in PCL and investigated by FNC, combined to ancillary techniques, is reported. METHODS: Twenty-one lymph node FNC from as many PCL patients were retrieved; 17 patients had mycosis fungoides (MF) and 4 a primary cutaneous B-cell lymphoma (PBL). In all cases, rapid on site evaluation (ROSE) was performed and additional passes were used to perform flow cytometry (FC), immunocytochemistry (ICC) and/or polymerase chain reaction (PCR) to assess or rule out a possible clonality of the corresponding cell populations. RESULTS: FNC combined with FC, ICC, and PCR identified 12 cases of reactive, non specific, hyperplasia (BRH), 4 dermatopathic lymphadenopathy (DL), 4 lymph nodal involvement by MF and 1 lymph nodal involvement by cutaneous B-cell lymphoma. CONCLUSIONS: FNC coupled with ancillary techniques is an effective tool to evaluate lymph node status in PCL patients, provided that ROSE and a rational usage of ancillary techniques is performed according to the clinical context and the available material. The method can be reasonably used as first line procedure in PCL staging and follow up, avoiding expensive and often ill tolerated biopsies when not strictly needed.

Clonal B-cell populations in non-lymphomatous processes have been sporadically reported in enlarged reactive lymph nodes and mucosa-associated lymphoid cell populations. These generally small clones are considered non-malignant proliferations of B-lymphocytes determined by an abnormal response to bacterial or viral antigen stimulation. In cases reported in literature, clonality was detected by light chain assessment and or by polymerase chain reaction (PCR) analysis of immunoglobulin heavy chain (IgH) gene in histologically and clinically proven non lymphomatous processes. In this study the clinical, cytological, phenotypical and pathological features of three HIV patients in which non-lymphomatous clonal B-cell populations detected in enlarged lymph nodes are reported. All the patients complained for later cervical lymph nodes enlargement, positive at the FDG-positron emission tomography scan. Fine needle cytology, coupled with flow cytometry showed atypical lymphoid cell proliferations and kappa (2 cases) or lambda (1 case) light chain restriction. Reactive, non lymphomatous nature of these processes were then proven by histological control in two cases and by clinical follow-up in the last one; corresponding clinical and pathological aspects are discussed. Clonal B-cell populations in non-lymphomatous processes can sporadically occur in enlarged reactive lymph nodes in immunodeficiency as well as in autoimmune processes. Awareness of the phenomenon and attention should be paid in the evaluation of corresponding pathological features and in the clinical management of corresponding patients.

SOMMARIO L’adenocarcinoma con ghiandole a citoplasma schiumoso (foamy-gland) è una variante istologica all’adenocarcinoma duttale del pancreas (ADP) con aspetto simil-benigno. La diagnosi differenziale con patologie benigne su prelievo ago-aspirato ottenuto per via endoscopica eco-guidata può pertanto risultare difficile. Riportiamo un caso di ADP foamy-gland ottenuto correlando le in ad una sospettato in strisci Diff Quick, diagnosticata in preparazione cell-block e confermata da tecniche di biologia molecolare. SUMMARY The foamy gland pattern (FGP) may impart to pancreatic ductal adenocarcinoma (PDA) a deceptively benign appearance. Thus, its diagnosis on FNA is challenging. A case of PDA with FGP, that was suspected on Diff Quik smears, diagnosed on cellblock preparation and confirmed by ancillary stains and molecular techniques, is here reported. The diagnostic interpretation of foamy atypical cells on direct smears may benefit from cell block preparation and from ancillary techniques.

The human immunoglobulin heavy-chain (IGH) locus at chromosome 14q32 is frequently involved in different translocations of non-Hodgkin lymphoma (NHL), and the detection of any breakage involving the IGH locus should identify a B-cell NHL. The split-signal IGH fluorescence in situ hybridization-chromogenic in situ hybridization (FISH-CISH) DNA probe is a mixture of 2 fluorochrome-labeled DNAs: a green one that binds the telomeric segment and a red one that binds the centromeric segment, both on the IGH breakpoint. In the current study, the authors tested the capability of the IGH FISH-CISH DNA probe to detect IGH translocations and diagnose B-cell lymphoproliferative processes on cytological samples.Fifty cytological specimens from cases of lymphoproliferative processes were tested using the split-signal IGH FISH-CISH DNA probe and the results were compared with light-chain assessment by flow cytometry (FC), IGH status was tested by polymerase chain reaction (PCR), and clinicohistological data.The signal score produced comparable results on FISH and CISH analysis and detected 29 positive, 15 negative, and 6 inadequate cases; there were 29 true-positive cases (66%), 9 true-negative cases (20%), 6 false-negative cases (14%), and no false-positive cases (0%). Comparing the sensitivity of the IGH FISH-CISH DNA split probe with FC and PCR, the highest sensitivity was obtained by FC, followed by FISH-CISH and PCR.The split-signal IGH FISH-CISH DNA probe is effective in detecting any translocation involving the IGH locus. This probe can be used on different samples from different B-cell lymphoproliferative processes, although it is not useful for classifying specific entities. Cancer (Cancer Cytopathol) 2012;. © 2012 American Cancer Society.

Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) is an established procedure in lung cancer (LC) staging and in the diagnosis of mediastinal masses. Most of the experiences reported refer to single specialized centers where dedicated teams of endoscopists and pathologists perform the procedure. We report the EUS-FNA experience of a cooperation group involving clinicians and cytopathologists from three hospitals. Fifty-seven consecutive EUS-FNA of mediastinal nodes in LC patients, eight mediastinal and two subdiaphragmatic masses were collected in 3 years. EUS-FNA was performed by two endoscopists and three experienced pathologists. On-site evaluation was performed in all cases by the three cytopathologists. Lymph node negative cases underwent surgery, which confirmed the cytological diagnoses but also detected two false negatives. Four of the 10 EUS cytological diagnoses of mediastinal and subdiaphragmatic masses were histologically confirmed. All EUS diagnoses were blindly reviewed by three pathologists to assess intra and interpersonal reproducibility. FNA-EUS diagnoses were: 10 inadequate (17%), 10 negative (17%), 4 suspicious (7%) and 33 positive (59%). Diagnoses of mediastinal and subdiaphragmatic masses were: relapse of lung carcinoma (3), mesenchimal tumor not otherwise specifiable (3), gastrointestinal stromal tumor (GIST) (1), esophageal carcinoma (2) and paraganglioma (1). The sensitivity attained was 85% and the specificity 100%; revision of the slides demonstrated a significant diagnostic reproducibility of the three cytopathologists (P < 0.5). The sensitivity and specificity attained were similar to those reported in the literature suggesting that experienced cytopathologists and endoscopists from different institutions can employ the same procedure reaching comparable results.

BACKGROUND: Pheochromocytomas/paragangliomas are rare tumors originating from neuroectodermic cells, which metastasize in only 10% of cases. CASE: A case of cutaneous metastasis of an extramedullary paraganglioma was diagnosed by fine needle aspiration cytology (FNAC). A 39-year-old woman complained of a recently arisen subcutaneous nodule located on the scalp. Two years before she had suffered from a sporadic extraadrenal paraganglioma located on the sacrum, and it had been evaluated by FNAC. Smears were highly cellular and monomorphous; the cells were uniform and mainly isolated with round or ovoid nuclei, dense chromatin and inconspicuous nucleoli, if any. The cytoplasm was ill defined, clear or granular. Oval-spindle-shaped cells and occasional cells with larger nuclei were also observed. Abundant fibrillar material was present in the background, intermingled with the surrounding cells. The immunocytochemical staining performed on cell block sections showed negativity for CK20, HMB45 and LCA and positivity for chromogranin and S100. The cytologic diagnosis was a neuroendocrine tumor, consistent with a metastasis from the former sacral paraganglioma. CONCLUSION: On conventional samples the cytologic features of paraganglioma are typical enough to allow a cytologic diagnosis of a neuroendocrine tumor; this neoplasm should be considered in the differential diagnosis, even in such unusual locations, because of its unpredictable biologic behavior.

Primary non-Hodgkin lymphoma of the oral cavity (OCL), represent less than 3% of all the NHL. pNHL may be clinically indistinguishable from other tumoral and non-tumoral lesions of the same area and the microscopical diagnoses may be hampered by the morphological overlap with reactive processes. An accurate pre-operative diagnosis should be mandatory because of the difficulties of a surgical biopsy and of a complete excision of the corresponding lesions. Fine needle cytology (FNC) has been successfully utilized in the preoperative diagnosis of oral cavity masses and NHL as well. The aim of this study has been to review the cytopathological features of a series of OCL. Materials and Methods:The series accounts for 10 cytological cases of OCL; one case of myeloma and one of florid lymphoid reactive hyperplasia were added to the series too. Corresponding lesions were from palatal soft tissue (2 cases); palatal bone (4 cases); floor of the mouth (1 case), vestibules (2 cases) and gums (3 cases). In all the cases, a traditional FNC was performed, in 7 cases, a second FNC was performed to prepare additional smears for immunocytochemistry (ICC) (2 cases), using antibodies CD3 and CD20 or suspended in PBS and used for flow cytometry (FC) in 5 cases. Cytological diagnoses were rendered on the basis of cytological features only (5 cases), or combined to ICC (2 cases) and/or FC (5 cases). All the diagnoses were histologically controlled. Results: Clinical presentation was generally represented by sub-mucosal masses which bulged in the oral cavity. In all the cases cytological smears showed a monomorphous lymphoid cell population; corresponding cells had nuclear atypia (enlargement, dispersed chromatin, membrane irregularity and 109 poster presentations Downloaded by: International Academy of Cytology 85.18.242.249 - 6/20/2013 1:42:07 PM large nucleoli) in 6 cases and more bland appearance in the 5 cases. One case showed a proliferation of atypical plasma-cells including binucleated and immature cells. Cytological diagnoses were suspect NHL in 7 cases including a case subsequently diagnosed as reactive hyperplasia. OCL was diagnosed in 4 cases and myeloma in 1 case. All the lesions were histologically diagnosed as NHL in 10 cases, myeloma (1) and florid lymphoid reactive hyperplasia (1). Conclusion: The cytological diagnosis of OCL is generally difficult, nonetheless a preoperative cytological diagnosis should be advisable to differentiate the corresponding masses from other tumoral and nontumoral processes, to avoid unnecessary extensive surgery and to sped up the therapeutic procedures

A case of ectopic cervical thymoma (ECT) in which fine needle cytology (FNC) and flow cytometry (FC) have orientated the cytologic diagnosis, is described. CASE: A 57-year-old woman underwent FNC of a right latero-cervical nodule. The smear showed a dispersed lymphoid-cell population; therefore, a second FNC was used for FC and to prepare a cell block. Smears were highly cellular. Cells were medium or large sized, with scanty cytoplasm and nuclei with dispersed chromatin; large cells showed evident nucleoli. Immunohistochemistry on additional smears were positive for CD45RO and Ki67 in most of the cells, and negative for CK pan, CD20, thyreoglobulin and calcitonin. FC showed the following phenotype: CD2/CD3/CD7 = 67%, CD10 = 61%, CD4/CD8 = 62%. CD19 and light chains were not expressed. A diagnosis of T-cell lymphoid proliferation was made and ECT was suggested; histological diagnosis was cervical ectopic benign type B1 thymoma. CONCLUSION: FC may support the FNC diagnosis of ECT because of the specific phenotype of lymphoid cells showing the profile of "polyclonal" (CD2/CD3/CD7+) and thymic T-cells (CD10+, CD4/CD6+). FNC and FC may suggest the diagnosis of ECT even in the absence of detectable epithelial cells.