William R. Geddie

EGFR gene status in cytological samples of nonsmall cell lung carcinoma: controversies and opportunities.

In nonsmall cell lung cancer (NSCLC), the development and clinical application of tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) has required the investigation of EGFR status by gene copy number and/or mutation analysis. This review aimed to present the current knowledge of the use of cytological specimens for EGFR testing in lung cancer.

A microfluidic device for simultaneous electrical and mechanical measurements on single cells

This paper presents a microfluidic device for simultaneous mechanical and electrical characterization of single cells. The device performs two types of cellular characterization (impedance spectroscopy and micropipette aspiration) on a single chip to enable cell electrical and mechanical characterization. To investigate the performance of the device design, electrical and mechanical properties of MC-3T3 osteoblast cells were measured. Based on electrical models, membrane capacitance of MC-3T3 cells was determined to be 3.39±1.23 and 2.99±0.82 pF at the aspiration pressure of 50 and 100 Pa, respectively. Cytoplasm resistance values were 110.1±37.7 kΩ (50 Pa) and 145.2±44.3 kΩ (100 Pa). Aspiration length of cells was found to be 0.813±0.351 μm at 50 Pa and 1.771±0.623 μm at 100 Pa. Quantified Youngs modulus values were 377±189 Pa at 50 Pa and 344±156 Pa at 100 Pa. Experimental results demonstrate the devices capability for characterizing both electrical and mechanical properties of single cells.

Electrodeformation for single cell mechanical characterization

This paper presents the use of electrodeformation as a method for single cell mechanical characterization in which mechanical properties of SiHa and ME180 cells (two cervical cancer cell lines) were quantified. Cells were directly placed between two microelectrodes with a rectangular ac electric field applied, and cell deformation was recorded under certain experimental conditions. Numerical simulations were performed to model cell electrodeformation based on the Maxwell stress tensor formulation. In these simulations, effects of cell electrical property variations on their electrodeformed behavior were investigated. By comparing the measured morphological changes with those obtained from numerical simulations, we were able to quantify Youngs modulus of SiHa cells (601 ± 183 Pa) and ME180 cells (1463 ± 649 Pa). These values were consistent with Youngs modulus values (SiHa: 400 ± 290 Pa and ME180: 1070 ± 580 Pa) obtained from conventional micropipette aspiration.

“The petals and thorns” of ROSE (rapid on‐site evaluation)

The small samples obtained by these procedures can be used for diagnosis based on morphologic criteria alone and also, in many instances, for special ancillary studies, including molecular analysis. The inherent challenges involved in handling and processing limited samples in such a way as to allow multiple studies have triggered changes in the preanalytical phase and generated new protocols to maximize tissue/cell retrieval. 5 ROSE has been advocated as an effective way to ensure that samples are handled properly for morphological analysis, and that they meet all preanalytical requirements for specific diagnostic tests. Emerging novel molecular diagnostic technologies in current pathology practice have reinforced the essential role of ROSE in allowing pathologists to accurately divide diagnostic cytological material into small aliquots. This procedure ensures that sufficient quantities of cells of adequate quality are obtained to permit a complete diagnostic workup. Ultimately, this will translate into an appropriate treatment plan. For CT-guided and endoscopicguided procedures, ROSE has become increasingly more relevant because of the higher (although still low) rate of complications and the intrinsic risks of these procedures when compared with FNA of superficial lesions. A recent trial has concluded that ROSE of transbronchial aspirates from hilar and mediastinal lymph nodes enables clinicians to avoid additional biopsy without a loss in the diagnostic yield and reduces the complication rate of bronchoscopy. 6

Detection of EGFR and KRAS mutations in fine-needle aspirates stored on Whatman FTA cards: is this the tool for biobanking cytological samples in the molecular era?

The aims of this study were to compare the quality of DNA recovered from fine‐needle aspirates (FNAs) stored on Whatman FTA cards with that retrieved from corresponding cell blocks and to determine whether the DNA extracted from the cards is suitable for multiple mutation analyses.

Characteristics of Apparently False-Negative Digene Hybrid Capture 2 High-Risk HPV DNA Testing

This study characterized cases with a negative high-risk Hybrid Capture 2 (HRHC2; Digene, Gaithersburg, MD) test result with concurrent or follow-up biopsy-confirmed high-grade cervical intraepithelial neoplasia (CIN 2/3). From 2,306 HRHC2 tests, 10 negative results were identified with CIN 2/3 (false-negative rate, 4.5%). The majority of the patients had abnormal colposcopic findings and high-grade squamous intraepithelial lesion (HSIL) shown by concurrent cytologic examination, although with few abnormal cells. No trend was evident in the location of the dysplastic epithelium or overall lesion size. In 4 tests, the relative light units over cutoff was more than 0.4 but less than 1.0, suggesting that low quantities of human papillomavirus (HPV) DNA were present in the sample. The negative predictive value for HRHC2 testing may be compromised when the copy number of the HPV DNA is low, and a negative HRHC2 test result may be falsely negative in patients with abnormal colposcopic findings or concurrent cytologic findings showing HSIL.

Targeted use of fluorescence in situ hybridization (FISH) in cytospin preparations: results of 298 fine needle aspirates of B-cell non-Hodgkin lymphoma.

Fluorescence in situ hybridization (FISH) results from fine needle aspirates (FNA) of B‐cell non‐Hodgkin lymphomas (NHLs) were reviewed to 1) investigate the value added by using specific gene rearrangement probes to lymphoma diagnosis, prognosis, and subtyping; and 2) evaluate the prevalence of cytogenetic alterations other than specific translocations.

Cyto-histologic agreement in pathologic subtyping of non small cell lung carcinoma: review of 602 fine needle aspirates with follow-up surgical specimens over a nine year period and analysis of factors underlying failure to subtype.

BACKGROUND The differential therapeutic efficacy and toxicity of targeted therapies has made subtyping of non-small lung cancer (NSCLC) mandatory. This study aimed to review the accuracy of NSCLC subtyping using lung fine needle aspirates (FNAs) in two periods (before and after the introduction of targeted therapy), checking the reasons for failure and the impact of the use of immunohistochemistry (IHC). METHODS An electronic search retrieved all NSCLC FNAs with a corresponding surgical specimen from 2001 to 2009. NSCLC, NOS (not otherwise specified) cases from 2005 to 2009 (after targeted therapy) were reviewed to determine reasons for failure in subtyping and to further subtype based solely on cytomorphology. The number of cases in which IHC was performed and the antibodies used were also recorded. RESULTS Cytohistological agreement of 602 lung FNAs (341 adenocarcinomas, 93 squamous cell carcinomas and 168 NSCLC, NOS) was achieved in 93.80%. There was a significant decrease in the percentage of cases not subtyped in the period after the introduction of targeted therapy (35.07% versus 24.57%). Final percentage of cases not subtyped after morphological review was 17.03%. IHC was performed in 157 cases, with an increased use in recent years. The number of antibodies did not influence the overall success in subtyping and an average of 3 markers was used. Most frequent antibodies used were TTF-1, CK7, high molecular weight keratin and p63. More than half of cases not subtyped even after IHC corresponded to poorly or undifferentiated neoplasms in the surgical specimens. For the NSCLC, NOS which IHC was not performed, a cell block was produced in 106 cases (75.71%). Review of the cell block slides from 2005 to 2009 showed that the majority (70.7%) had rare, few or no tumor cells. CONCLUSIONS Specific subtyping can be achieved in a high proportion of lung FNAs with high accuracy. The percentage of NSCLC, NOS has significantly decreased in recent years together with a trend for an increased use of IHC as well as increased number of cell blocks produced. An average of 3 IHC markers was used for subtyping and the number of markers did not influence the overall subtyping.

The use of FTA cards for preserving unfixed cytological material for high-throughput molecular analysis

Novel high‐throughput molecular technologies have made the collection and storage of cells and small tissue specimens a critical issue. The FTA card provides an alternative to cryopreservation for biobanking fresh unfixed cells. The current study compared the quality and integrity of the DNA obtained from 2 types of FTA cards (Classic and Elute) using 2 different extraction protocols (“Classic” and “Elute”) and assessed the feasibility of performing multiplex mutational screening using fine‐needle aspiration (FNA) biopsy samples.

The role of fine-needle aspiration in the diagnosis of thyroid lymphoma: a retrospective study of nine cases and review of published series

Aims To review the clinicopathological, cytomorphological and immunophenotyping data from new cases and published series of thyroid lymphoma diagnosed by fine-needle aspiration (FNA), in order to identify useful diagnostic features. Methods Cases from 1988 to 2009 with an FNA diagnosis of thyroid lymphoma were selected from hospital records. An electronic MEDLINE(R) and EMBASE search retrieved published series from 1980 to 2009. Available clinical, cytomorphological and immunophenotyping data from all cases were collected. In our cases, cytology slides and available surgical specimens were also reviewed. Results There were nine cases from eight of our patients, and 70 reviewed cases from eight series with at least four patients each. The most common presentation was a rapidly enlarging thyroid mass. Average patient age was 61 years in reviewed cases and 72 years in our cases. Large-cell lymphoma was the predominant subtype, revealing relatively monotonous populations of large, abnormal lymphoid cells. One of our cases, later diagnosed as marginal zone lymphoma, showed small lymphocytes with plasmacytoid features. Immunoprofiling information was available in five of our cases (three by immunocytochemistry and two by laser scanning cytometry) and in 34 reviewed cases (22 by immunocytochemistry, six by flow cytometry, and six by flow cytometry or immunocytochemistry). Conclusions Cytological diagnosis of thyroid lymphoma requires careful analysis of morphological, clinical and immunophenotypic information. The presented data suggest certain helpful features: a fast-growing nodule in an elderly patient, a monotonous population of large abnormal cells in a background of lymphoglandular bodies, a predominant population of plasmacytoid lymphocytes, and immunophenotyping demonstrating light chain restriction.