Liquid-Based Cytology: A 25-Year Bridge between the Pap Smear and Molecular Cytopathology

Liquid-based cytology (LBC), was first applied to gynecological cytology almost twenty-five years ago (1). A few years before, the conventional Pap smear, after decades of unquestionable success proven by the drop of morbidity and mortality due to cervical carcinoma in the countries that had adopted it in national screening programs, met some criticism in international media. Because of a reported rate of false negatives and some inefficiencies, the Pap test was openly discussed and criticized on journals that were not usually devoted to specific scientific issues (2,3). The following and timely introduction of the LBC aimed to improve the efficiency of the gynecological cytology through procedure standardization, sample quality improvement, screening support and speeding-up, quality controls, as well as by storing the residual material for possible repetitions and/or the application of ancillary techniques. Whereas LBC seems to be neither more sensitive nor more specific than the conventional Pap test, mainly in the detection of high grade cervical intraepithelial neoplasia (4), overall it achieved the above reported goals, being its additional costs compensated by reported advantages. As consequence, LBC has replaced the conventional Pap smear in many labs in industrialized countries and promises to survive the era of cervical HPV testing and vaccination. Since its introduction, many labs started to apply LBC to exfoliative and aspiration non-gynecological cytology, aiming to exploit the same advantages on different samples. In fact, LBC is well suited to meet the requirements of ancillary techniques and their routine application in pathology labs, with specific reference to procedure standardization, purification and storage of diagnostic material. As a matter of fact, method standardization and the opportunity to store cells are a significant benefit of non-gynecologic LBC, which can be used in different organs, for different applications. Whereas the advantages of gynecological LBC have been immediately obtained on respiratory cytology (5), the same features of LBC have limited, in some cases, the diagnostic criteria typical of traditional cytology. For instance, the additional value of collected material and the time saved from the cytological assistance, mainly in CT, EUS, and EBUS-guided and other time-consuming FNA techniques, may hamper or even exclude the possibility to perform rapid on-site evaluation (ROSE) and the immediate repetition of inadequate samples. Moreover, at diagnostic level, the clean and purified background that improves and enhances cellular details in the gynecologic LBC or in bronchial washing-brushing, in thyroid and breast FNAs bereaves or reduces colloid and myoepithelial cells, as well as stroma fragments, which represent additional diagnostic criteria for goiter and fibroadenoma, respectively. Nonetheless, beyond the microscopic diagnostic criteria, procedure standardization and cell storage is the great advantage of non-gynecologic LBC that can be exploited in different cytological fields for different requests and applications. In this issue of Acta Cytologica, readers are provided with a comprehensive and critical overview of LBC applied to different non-gynecologic samples and different technical procedures. In the article by Ren et al. (6), basic LBC technical procedures and cytological features of different samples of exfoliative and aspirative cytology are clearly and synthetically reported, providing a LBC taxonomic handout. Remarkably, some thin-layer (TL)-specific cytological features that highlight their differences from conventional smears (CS), such as lymphocytes artificial aggregation or fragmentation of cohesive epithelial cells, are clearly reported (6) and the possible final reconversion of residual material in cell-blocks is also described. Breast FNA has been significantly reduced after the introduction of core needle biopsy; in addition to the impossibility to distinguish in-situ from invasive tumors or to correct diagnose papillary lesions, the main limitation of breast FNA is the operator dependency, and LBC provides the opportunity to overcome this latter limitation. In the article by Gerhard and Schmitt (7), specific cytological criteria and diagnostic performances of breast LBC are analytically reported showing that breast LBC sensitivity and specificity is equal to or, in some cases, even higher than CS values (7). The diagnostic “equality” between LBC and CS is overcome by the advantages offered by LBC in terms of antigen preservation, and the good reproducibility of HER-2 status by FISH or CISH in TL and histological controls. As far as thyroid FNA is concerned, follicular lesions of undetermined significance (FLUS) may be considered the Achilles’ heel of the otherwise most successful thyroidal FNA. In the article by Rossi et al (8), a comprehensive overview on the usage of HBME and galectin-3 on FLUS is reported, where LBC proves to be an excellent tool for this purpose. With reference to molecular procedures, LBC has been conveniently used on thyroidal FNAs for tests that evaluate a wide panel of mutations (9,10). Moreover, the next generation sequencing procedures that promise to further increase diagnostic sensitivity and specificity of thyroidal FNA, will probably use routine LBC samples too (11). As reported above, LBC may require different diagnostic criteria from those utilized on CS, and this is the case of salivary gland tumors. In the article by Rarick et al (12), LBC diagnostic criteria for salivary gland tumors are exhaustively described. Once more, LBC advantages may be exploited when FNA is performed by non-cytopathologists and liquid suspended cells may be alternatively used for LBC or cell blocks when ROSE is performed (10). Micronuclei (MN) are chromosome fragments or whole chromosomes that are excluded from the nucleus during mitosis. MN can be identified by the same nuclear staining and used to evaluate chromosomal instability in cancerous and pre-cancerous lesions. MN evaluation of exfoliated buccal cells has been performed since the eighties (13) mainly on people exposed to different environmental genotoxic agents. Ramos et al. (14) studied MN on exfoliated buccal cells in workers exposed to carcinogenic agents and demonstrated that LBC is an excellent tool for MN evaluation (14). Lung cytology has been the first non-gynecological application of LBC (4). Because of the similarities that bronchial washing and brushing share with gynecological cytology samples, LBC lung cytology has exploited the same advantages (5,15). In the article by Michael and Bedrossian (15), a detailed and analytical overview of LBC lung cytology is reported, including the evaluation and cost comparison of both Thin-prep and Tripath. Additional advantages of LBC lung cytology include the possible application of almost all the ancillary techniques; in the article by Bellevicine et al (16) the authors confirmed evidences generated by a previous validation studies (17). They reported the experience of an academic centralized laboratory demonstrating that LBC is suitable for EGFR analysis to select patients for targeted therapy in lung cancer. (16). In fact, in their practice EGFR testing performance features on LBC, such as the rates of sample adequacy and and of mutant, overlapped with those generated by direct smears. (17) Finally, the article by Abedi-Ardekani and Vielh (18) provides an in-depth analysis by summarizing and critically reviewing the most important applications of molecular techniques to LBC (18). In conclusions, non-gynecologic LBC has an increasingly important role in diagnostic cytopathology. LBC has a similar or better diagnostic accuracy than CS, provided that specific diagnostic criteria are applied. The possibility of storing additional material, useful for molecular procedures, provides LBC a great advantage that promises to be further exploited by the next-generation sequencing technology, and promotes LBC as a reliable alternative tool to surgical or core needle biopsies in cancer diagnosis, prognosis and prediction

Multiple metachronus proliferative fasciitis occurring in different anatomic regions: a case report and review of the literature.

Proliferative fasciitis is a benign lesion that usually has a self-limited course and rarely recurs after excision. In the literature, the multifocal occurrence of PF in different anatomic sites has not been reported so far. In this report, we describe the clinical case of a 30-year-old woman with two metachronous proliferative fasciitis occurring firstly in the orbit and, after 18 months, in the forearm; we also review the available literature on this topic, outlining guidelines for therapy and the follow-up of these patients

Management of Cytological Material, Pre-Analytical Procedures and Bio-Banking in Lymph Node Cytopathology

The range of pathologies that Lymph Node (LN) Fine-Needle Cytology (FNC) may deal with is extremely wide and ancillary techniques, in addition to traditional smears, are generally required to reach reliable cytological diagnoses. For this purpose, in the pre-analytical phase of LN-FNC, using the most effective vials, fixatives and supports is essential, since they may perform differently with different ancillary techniques, and even in different pathologies. Moreover, storing part of the cytological material may be useful or necessary for molecular testing. The main difficulties concern the generally small size of the sample and the different ways of acquisition of LN-FNC. Therefore, the pre-analytical phase is extremely important for LN-FNC. This study investigates the management of LN-FNC material, vials, technical supports and main ancillary techniques in order to assess their optimal application, taking into account the different diagnostic needs and cell storage. This article is protected by copyright. All rights reserved

Fine-needle aspiration biopsy and flow cytometry immunophenotyping of lymphoid and myeloproliferative disorders of the spleen.

BACKGROUND: Flow cytometry (FC) is a useful adjunct to fine-needle aspiration biopsy (FNAB) in the evaluation of lymphoproliferative disorders. The application of FC to FNAB of the spleen (sFNAB) is reported. METHODS: Flow cytometry was performed on 18 sFNAB collected over 3 years. The series comprised 10 cases of non- Hodgkin lymphomas (NHL), 2 cases insufficient for diagnosis, 2 cases of reactive hyperplasia (RH), and 4 cases of myeloid metaplasia (MM). FNAB was performed under ultrasound guidance using a 22-gauge needle. One or two passes were sufficient to prepare a conventional smear that was immediately evaluated to select the cases studied and to prepare a cell suspension for FC. The following fluoresceinated antibodies were used: CD3, CD19/kappa/lambda, FMC7/CD23/CD19, Bcl-2, and CD13/HLA-DR. In six cases, cytospins were also prepared for immunocytochemistry and were tested for CD20 (L26), CD45Ro, and kappa and lambda light chain expression. RESULTS: Flow cytometry contributed to the diagnosis of all cases of NHL by assessing light chain restriction. The specific subtype was also diagnosed by CD19/CD5 and CD 19/CD10 coexpression in two cases. Flow cytometry quantified the percentage of myeloid cells in MM cases and contributed to the cytologic diagnosis showing a polyclonal light chain expression in RH cases.Immunocytochemistry was effective and concordant in four cases. Patients tolerated the sFNAB well and no complications were reported. Cytologic and FC diagnoses were confirmed by follow-up and by histologic evaluation in cases in which splenectomy was performed for therapeutic purposes. CONCLUSION: Flow cytometry applied to sFNAB corroborates the cytologic diagnosis in lymphoid and myeloproliferative disorders of the spleen and allows therapeutic decisions avoiding splenectomy

Surgical management of cervico-mediastinal goiters: Our experience and review of the literature

We analyze and discuss the clinical presentation, the diagnostic procedures and the surgical technique in relation to post-operative complications and results in cervico-mediastinal thyroid masses admitted in Thoracic Surgery Unit of AOU Second University of Naples from 1991 to 2006 and in Thoracic Surgery Unit of AOU "S. Giovanni di Dio & Ruggi D'Aragona" of Salerno over a period of 3 years (2011-2014)

Immunoglobulin heavy-chain fluorescence in situ hybridization-chromogenic in situhybridization DNA probe split signal in the clonality assessment oflymphoproliferative processes on cytological samples.

BACKGROUND: 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. METHODS: 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 AQ2 (FC), IGH status was tested by polymerase chain reaction (PCR), and clinicohistological data. RESULTS: 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. CONCLUSIONS: 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

Kidney Transplant Modifies the Architecture and Microenvironment of Basal Cell Carcinomas

Background/Aims: Basal cell carcinoma (BCC) is a frequent type of nonmelanoma skin cancer, which shows a greater prevalence in kidney-transplanted (KT) patients than in the general population. The study of this tumor in KT patients may allow us to understand the influence of the tumor inflammatory microenvironment on cancer behavior, and to design new image analysis methods to determine prognosis and apply personalized medicine. The major hypothesis of the present work is that antirejection drugs, by modifying the B-cell/T-cell balance, induce measurable differences in tumoral cell microarchitecture and in the inflammatory microenvironment in KT patients compared to nontransplanted controls. Methods: In this retrospective study in an Italian cohort including 15 KT patients and 15 control subjects from the general population who developed BCC, we analyzed tissue microarchitecture and inflammatory infiltrates of BCC using state-of-the-art nonlinear image analysis techniques such as fractal dimension and sample entropy of internuclear distances. Results: KT patients showed a nonsignificant trend to a greater number of nuclei in the basal cell layer compared to non-KT controls and subtle changes in the intact skin compared to controls. Similarly, the number of mitoses per unit length was almost doubled in the patients with KT compared to controls. However, when the number of mitotic cells was normalized by the total number of cells in the basal layer (mitotic index), these differences were not significant, although a clear trend was still present. Finally, KT patients showed a nonsignificant trend to an increased ­density of inflammatory cells close to the tumoral cell layer. When considering the intact skin, this difference was significant, with a 70% increase in the density of inflammatory cells. Conclusion: Data comparing the microarchitecture of BCC in normal subjects and KT patients are scanty, and the present study is the first to use nonlinear image analysis techniques to this aim. The observed differences underscore the relevance of T-cell suppression in cancer behavior. These data suggest that BCC develops in treated patients with specific biological characteristics which should be further analyzed in terms of therapeutic response

Randomized comparison of power Doppler ultrasound-directed excisional biopsy with standard excisional biopsy for the characterization of lymphadenopathies in patients with suspected lymphoma.

PURPOSE: The sensitivity of lymph node excisional biopsy requires validation. Power Doppler ultrasound (US) helps predict the malignant status of lymphadenopathies. We used power Doppler US to select for biopsy the lymph node most suspected of malignancy. PATIENTS AND METHODS: One hundred fifty-two patients having lymphadenopathies with clinical suspicion of lymphoma were divided into two well-matched groups and randomly assigned to undergo either standard or power Doppler US-directed lymph node excisional biopsy. RESULTS: Histology showed a malignancy in 64% of patients in the standard group (lymphoma, 49 patients; carcinoma, two patients) and in 87% of patients in the US-assisted group (lymphoma, 62 patients; carcinoma, one patient). There were significantly fewer biopsy-related complications in the assisted group than in the standard group. During the follow-up of the patients with lymph nodes reported as being reactive, 14 of 29 patients in the standard group were rebiopsied and were found to have lymphoma (13 patients) or carcinoma at the subsequent lymph node histology, whereas none of the patients in the assisted group (nine patients) required a second biopsy. Thus, biopsy provided false-negative results for malignancy in 21% of patients affected by lymphoma in the standard group and ever in the assisted group (P <.01). CONCLUSION: Power Doppler US is an accurate tool for screening lymphadenopathies to be removed by excisional biopsy in patients with suspected lymphoma