The use of the 'borderline' category in the reporting of cervical cytopathology in the UK: results of a survey conducted under the aegis of the British Society for Clinical Cytology

Objective: To determine how the ‘borderline’ category was used by cytopathologists in the UK when reporting cervical smears. Methods: A questionnaire was sent by email to members of the British Society for Clinical Cytology. Results: There is wide variation in the use of the ‘borderline’ category in the UK but the majority of respondents (77.6%) used it when reporting smears that were either on the borderline between negative and low grade squamous dyskaryosis (‘borderline ?low grade’), or on the borderline between negative and high grade squamous dyskaryosis (‘borderline ?high grade’), or on the borderline between negative and glandular dyskaryosis ‘borderline ?glandular dyskaryosis’). A significant minority (15.7%), however, did not use ‘borderline’ when reporting smears that showed an abnormality that was possibly high grade squamous dyskaryosis. A majority (79.1%) of respondents thought that it would be useful to have separate reporting categories for ‘borderline ?low grade’ and ‘borderline ?high grade’. Conclusions: There is diversity in the use of the category ‘borderline’ in the UK. The proposed revised BSCC terminology with separate categories for borderline ?low grade, borderline ?high grades and borderline ? glandular dyskaryosis reflects the opinion of the majority of respondents to the questionnaire.     

Interlaboratory reproducibility of atypical squamous cells of undetermined significance report: a national survey

The study was aimed at assessing interlaboratory reproducibility in the reporting of cervical smears in the atypical squamous cells of undetermined significance (ASCUS) category. A set of 50 selected slides circulated among 89 laboratories, currently involved in population-based screening programmes for cervical cancer, which provided a diagnostic report according to four main reporting categories based on the 1991 Bethesda system. Interlaboratory agreement was determined according to kappa (K) statistics: overall and weighted K values were determined for each laboratory and for single reporting categories. The results showed a very low reproducibility for the ASCUS category. This finding supports the Bethesda system 1991 recommendation to limit the use of this reporting category and suggests that the clinical response to ASCUS reports should be decided locally, based on the observed positive predictive value for cervical intraepithelial neoplasia 2 or more severe lesions.     

Increased diagnostic accuracy of atypical glandular cells in cervical liquid‐based cytology using cell blocks

E. K. J. Risse, J. P. Holierhoek, E. M. Meijer‐Marres, E. Ouwerkerk‐Noordam and M. E. Boon Increased diagnostic accuracy of atypical glandular cells in cervical liquid‐based cytology using cell blocks Objective: The purpose of this study was to reduce the number of diagnoses of atypical glandular cells (AGC). Residual material from the cervical ThinPrep® samples (Hologic, Marlboruogh, MA, USA) was used for cell blocks (CB) and immunohistochemistry (IHC). Methods: In 2007 there were 87 patients (0.12% of tests) with AGC on liquid‐based cytology (LBC) in the Leiden Cytology and Pathology Laboratory (LCPL) using the Bethesda System 2001 (TBS). CB with IHC was used for 26 of these cases. The vials still containing the brush (Cervex‐Brush^® Combi) were placed in a shaker for 10 minutes to dislodge the material trapped between the bristles. The residual sampling fluid was used to prepare paraffin sections (Shandon Cytoblock^®) stained with Papanicolaou and immunostaining. Results: Four of five cases with AGC not otherwise specified (NOS) were diagnosed with CB/IHC as benign mimics (endometrium, tubal metaplasia, follicular cervicitis, microglandular hyperplasia) and one of four with AGC‐favour neoplasia (FN) (endocervical polyp). In one of five cases with AGC‐NOS and in two of seven with AGC‐FN, CIN3 was found on subsequent histological biopsy. Of six cases diagnosed as adenocarcinoma in situ (AIS) on LBC with CB/IHC the diagnosis was confirmed in four; one was adenocarcinoma and one glandular atypia. Of eight cases diagnosed as adenocarcinoma on cytology and CB/IHC, the diagnosis was confirmed in three. The other five cases were found to be one each of AIS, squamous cell carcinoma, CIN3, CIN2 with glandular atypia, and cervical endometriosis. Conclusions: By reducing the number of benign mimics of AGC, we achieved a high proportion (16/26; 61.5%) of neoplastic or preneoplastic lesions (glandular or squamous) on histological outcome potentially avoiding colposcopy. Histological biopsy verification by the gynaecologist is needed for final diagnosis of AGC‐FN, AIS and adenocarcinoma.     

Atypical squamous cells of undetermined significance: audit and the impact of potential litigation. Retrospective review of 682 cases

For quality assurance purposes, the frequency of 'abnormal' cytological diagnoses of the non-systematic National Cervical Cancer Screening Programme (NCCSP) was evaluated. In 1999, an unexpected high number of Class (Cl) III cases (i.e. atypical squamous cells of undetermined significance) was reported. The cytological and histological results were reviewed in order to detect a possible cause for this threefold increase. The abnormal Papanicolaou (PAP) smears examined by conventional methods from 1 January 1990 to 31 December 2002 were analysed. The smears of 682 cases diagnosed in 1999 with a Cl III category were reviewed in 2000 and correlated with the available histological diagnoses provided by the Central Department of Pathology. Of the 682 Cl III cases, 176 cases (26.1%) had no follow-up, 314 cases (46.0%) had repeat cytology and 192 cases (28.2%) an histological correlate corresponding to 90 (46.9%) benign lesions, 78 (40.6%) squamous intraepithelial lesions, two (1%) invasive cervical cancers (one squamous and one glandular). Twenty-two Cl III cases (11.5%) were histologically within normal limits. Retrospective smear review confirmed 330 Cl III diagnoses (48.3%), 127 cases (18.6%) were recategorized as Cl IIIG (i.e. atypical glandular cells of undetermined significance), 22 cases (3.2%) as Cl IIID (i.e. mild to moderate dysplasia) and six cases (0.9%) as Cl IVa (i.e. severe dysplasia and/or carcinoma in situ). A total of 197 original Cl III cases had to be reclassified in the Cl II category (28.9%), only two cases showing mild and moderate dysplasia on histology. Thus, 195 cases (28.6%) comprised cytological overdiagnoses. The Cl III category being, by definition, a delicate and often subjective diagnosis, all external influences such as pressure of litigation should be avoided to reduce cytological overdiagnoses as a result of an unnecessary 'fear-factor'.     

Expression of cytokeratin 20 in urine cytology smears: a potential marker for the detection of urothelial carcinoma

BACKGROUND: Urine cytomorphology is one of the oldest methods for screening and monitoring patients with transitional cell carcinoma (TCC). Sensitivity of urine cytology is relatively low. Ancillary techniques on urine sample may increase the sensitivity. AIM: To explore the utility of cytokeratin 20 (CK20) immunostaining in identifying malignant cells in urine cytology smears. MATERIALS AND METHODS: Fourteen cases each of confirmed TCC and benign urinary cytology along with five cases of atypical cells in urine were immunostained with a monoclonal CK20 antibody. Of 14 cases of TCC, 12 showed strong positive staining with the antibody. All benign cases were negative except for a few cases in which the umbrella cells were weakly to moderately positive. In all five cases of atypical urine cytology the atypical cells stained positive with the antibody. These cases were later confirmed as TCC on histopathology of bladder wall biopsy. CONCLUSION: CK20 is an important biomarker that can be used to identify TCC in urine cytology smears. It is particularly useful in those cases where malignancy cannot be confirmed by morphology alone.     

Triage of women with equivocal or low-grade cervical cytology results: a meta-analysis of the HPV test positivity rate

Consistent evidence underlines the utility of human papillomavirus (HPV) DNA testing in the management of women with equivocal cervical cytological abnormalities, but not in case of low-grade lesions. We performed a meta-analysis including studies where the high-risk probe of the Hybrid Capture-II is used to triage these two cytological categories. The triage test-positivity rate reflects the colposcopy referral workload.Data were pooled on the HPV test positivity rate in women with atypical squamous cells of undetermined significance (ASCUS/ASC-US) or low-grade squamous intraepithelial lesions (LSIL), derived from different cytological classification systems. The meta-analysis was restricted to studies, published between 1991 and 2007. A random-effect model was applied for meta-analytical pooling and the influence of covariates on the HPV positivity rate was analyzed by meta-regression. The variation by age was assessed within individual studies since age strata were not defined uniformly. On an average, 43% (95% CI: 40–46%) of women with ASCUS/ASC-US were high-risk HPV positive (range 23–74%). In women with LSIL, the pooled positivity rate was 76% (95% CI: 71–81%; range 55–89%). In spite of considerable inter-study heterogeneity, the difference in HPV positivity between the two triage groups was large and highly significant: 32% (95% CI: 27–38%). HPV rates dropped tremendously as age and cutoffs of test positivity increased. Other factors (cytological classification system, country, continent, collection method and year of publication) had no statistically significant impact, except in LSIL triage where HPV positivity was significantly lower in European compared to American studies. Women with LSIL, especially younger women, have high HPV positivity rates suggesting limited utility of reflex HPV triaging these cases. Research is needed to identify more specific methods to triage women with low-grade squamous cervical lesions.     

2D format for screening bacterial cells at the throughput of flow cytometry

We present a method for generating gel-based unordered 2D arrays of bacterial cells of a very high density, up to 10(5) cells per mm(2). Bacteria in a suspension are focused into a thin layer when the suspension and a dry gel matrix penetrate each other. Formation of a second gel from gel-forming components contained in the suspension results in immobilization of the cells. The immobilized cells stay alive and can repeatedly divide to produce microcolonies. The method provides for high-throughput screening and massively parallel analysis of individual cells in large populations, as well as for rapid isolation of rare clones.     

Implementation of a joint protocol for the management of thyroid nodules with a cytological diagnosis of follicular lesion of undetermined significance: experience in one hospital

Background: Follicular lesion of undetermined significance (FLUS) was introduced for fine needle aspiration (FNA) cytology in which there is insufficient evidence to classify the lesion as follicular neoplasm/suspicious of follicular neoplasm or suspicious for malignancy. The recommended management was repeat FNA and correlation with clinical and radiological data. In 2009 we started a joint clinicopathological protocol to improve management of FLUS, recommending follow‐up with repeat FNA at 6 months. The aim of this study was to report on the audit of results of this protocol. Methods: We reviewed the medical records of the patients with FLUS at a single hospital. Between 2007 and 2010 we found 135 cases with this diagnosis (3.6%). We only had long enough follow‐up information for the 95 patients that were included in the present study. Results: FLUS was diagnosed in 74 FNAs before protocol implementation (3.2%) and 61 FNAs after (4.2%), with follow‐up of 46 and 49 patients, respectively. Before 2009, 38/46 (82.6%) patients had surgical excisions, compared with 32/49 (65.3%): a significant reduction of 17% in the number requiring surgery (P = 0.05). We have also shown a reduction in the median time to surgery (11.9 versus 2.9 months). Despite the joint protocol, the FNA was only repeated in two patients. The histological diagnoses were similar in the two periods of time: 31.6% and 31.3% follicular adenomas; 13.1% and 3.1% (P = 0.2) papillary carcinoma (follicular variant). Conclusions: Implementation of a joint protocol reduced the number of surgical operations in patients with FLUS but in most cases FNA was not repeated as recommended. Excision was justified in one‐third of operated patients. Less than 15% of lesions were malignant, which is in accordance with previous reports in the literature.     

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-γ). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.     

Interobserver concordance in the assessment of features used for the diagnosis of cervical atypical squamous cells and squamous intraepithelial lesions (ASC‐US,ASC‐H,LSIL and HSIL)

G. Bigras, J. Wilson, L. Russell, G. Johnson, D. Morel and M. Saddik
Interobserver concordance in the assessment of features used for the diagnosis of cervical atypical squamous cells and squamous intraepithelial lesions (ASC‐US, ASC‐H, LSIL and HSIL) Objectives: Given the well‐known poor reproducibility of cervical cytology diagnosis, especially for atypical squamous cells of undetermined significance (ASC‐US) and low‐grade squamous intraepithelial lesion (LSIL), this study surveyed reproducibility in the assessment of individual cytomorphological features. Methods: One hundred and fifty cells or groups of cells, with a variety of morphological appearances, including normal cells, high‐grade squamous intraepithelial lesion (HSIL), LSIL, ASC‐US and ASC cannot exclude HSIL (ASC‐H), were precisely marked on 150 different liquid‐based cytological preparations. They were analysed by 17 observers who assessed 17 cytological features including nuclear features (chromatin texture, nuclear outline, nuclear shape, etc.), cytoplasmic features (cell shape, cytoplasmic staining, cytoplasmic clearing, etc.) and group characteristics (nuclear polarity, cellular density, etc.). A total of 43 350 data scores were collected in a database using a web‐based survey. Kendall’s W and relative entropy indexes were utilized to compute concordance indexes of respectively ordinal and nominal variables. Results: Nuclear features have significantly lower reproducibility (0.46) compared with other cytological features (0.59). The feature with least agreement is assessment of chromatin texture. A small but significant difference in concordance was found between two subsets of observers with different levels of experience. Conclusion: Most previous studies assessing reproducibility of cytological diagnoses show, at best, moderate reproducibility among observers. This study focused on agreement regarding the presence of constituent morphological features used to recognize dyskaryosis and various grades of squamous intraepithelial lesions. A map of reproducibility indexes is presented that highlights, for daily practice or teaching, the robustness of features used for cytological assessment, recognizing that diagnosis is always based on a combination of features.     

Tyrosine phosphorylation of 3BP2 is indispensable for the interaction with VAV3 in chicken DT40 cells

Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2) is known to play regulatory roles in immunoreceptor-mediated signal transduction. We have previously demonstrated that Tyr¹⁷⁴, Tyr¹⁸³ and Tyr⁴⁴⁶ in mouse 3BP2 are predominantly phosphorylated by Syk, and the phosphorylation of Tyr¹⁸³ and the Src homology 2 (SH2) domain of mouse 3BP2 are critical for B cell receptor (BCR)-induced activation of nuclear factor of activated T cells (NFAT) in human B cells. In this report, we have shown that Syk, but not Abl family protein-tyrosine kinases, is critical for BCR-mediated tyrosine phosphorylation of 3BP2 in chicken DT40 cells. Mutational analysis showed that Tyr¹⁷⁴, Tyr¹⁸³ and Tyr⁴²⁶ of chicken 3BP2 are the major phosphorylation sites by Syk and the SH2 domain of 3BP2 is critical for tyrosine phosphorylation. In addition, phosphorylation of Tyr⁴²⁶ is required for the inducible interaction with the SH2 domain of Vav3. Moreover, the expression of the mutant form of 3BP2 in which Tyr⁴²⁶ was substituted to Phe resulted in the reduction in BCR-mediated Rac1 activation, when compared with the case of wild-type. Altogether, these data suggest that 3BP2 is involved in the activation of Rac1 through the regulation of Vav3 by Syk-dependent phosphorylation of Tyr⁴²⁶ following BCR stimulation.     

Morphological analysis of circulating tumour cells in patients undergoing surgery for non‐small cell lung carcinoma using the isolation by size of epithelial tumour cell (ISET) method

V. Hofman, E. Long, M. Ilie, C. Bonnetaud, J. M. Vignaud, J. F. Fléjou, S. Lantuejoul, E. Piaton, N. Mourad, C. Butori, E. Selva, C. H. Marquette, M. Poudenx, S. Sibon, S. Kelhef, N. Vénissac, J. P. Jais, J. Mouroux, T. J. Molina, P. Vielh and P. Hofman
Morphological analysis of circulating tumour cells in patients undergoing surgery for non‐small cell lung carcinoma using the isolation by size of epithelial tumour cell (ISET) method Background and objective: Recurrence rates after surgery for non‐small cell lung cancer (NSCLC) range from 25 to 50% and 5‐year survival is only 60–70%. Because no biomarkers are predictive of recurrence or the onset of metastasis, pathological TNM (pTNM) staging is currently the best prognostic factor. Consequently, the preoperative detection of circulating tumour cells (CTCs) might be useful in tailoring therapy. The aim of this study was to characterize morphologically any circulating non‐haematological cells (CNHCs) in patients undergoing surgery for NSCLC using the isolation by size of epithelial tumour cell (ISET) method. Methods: Of 299 blood samples tested, 250 were from patients with resectable NSCLC and 59 from healthy controls. The presence of CNHCs was assessed blindly and independently by 10 cytopathologists on May‐Grünwald–Giemsa stained filters and the cells classified into three groups: (i) malignant cells, (ii) uncertain malignant cells, and (iii) benign cells. We assessed interobserver agreement using Kappa (κ) analysis as the measure of agreement. Results: A total of 123 out of 250 (49%) patients showed CNHCs corresponding to malignant, uncertain malignant and benign cells, in 102/250 (41%), 15/250 (6%) and 6/250 (2%) cases, respectively. No CNHCs were detected in the blood of healthy subjects. Interobserver diagnostic variability was absent for CNHCs, low for malignant cells and limited for uncertain malignant and benign cells. Conclusion: Identification of CTCs in resectable NSCLC patients, using ISET technology and according to cytopathological criteria of malignancy, appears to be a new and promising field of cytopathology with potential relevance to lung oncology.     

Transient proteasome inhibition as a strategy to enhance lentiviral transduction of hematopoietic CD34(+) cells and T lymphocytes: implications for the use of low viral doses and large-size vectors

The proteasome system restricts lentiviral transduction of stem cells. We exploited proteasome inhibition as a strategy to enhance transduction of both hematopoietic stem cells (HSC) and T lymphocytes with low dose or large-size lentiviral vectors (LV). HSC showed higher transduction efficiency if transiently exposed to proteasome inhibitor MG132 (41.8% vs 10.7%, p < 0.0001). Treatment with MG132 (0.5 μM) retained its beneficial effect with 3 different LV of increasing size up to 10.9 Kb (p < 0.01). We extended, for the first time, the application of proteasome inhibition to the transduction of T lymphocytes. A transient exposure to MG132 significantly improved lentiviral T-cell transduction. The mean percentage of transduced T cells progressively increased from 13.5% of untreated cells, to 21% (p = 0.3), 30% (p = 0.03) and 37% (p = 0.01) of T lymphocytes that were pre-treated with MG132 at 0.1, 0.5 and 1 μM, respectively. MG132 did not affect viability or functionality of HSC or T cells, nor significantly increased the number of integrated vector copies. Transient proteasome inhibition appears as a new procedure to safely enhance lentiviral transduction of HSC and T lymphocytes with low viral doses. This approach could be useful in settings where the use of large size vectors may impair optimal viral production.     

Cannabidiol as an emergent therapeutic strategy for lessening the impact of inflammation on oxidative stress

Oxidative stress with reactive oxygen species generation is a key weapon in the arsenal of the immune system for fighting invading pathogens and initiating tissue repair. If excessive or unresolved, however, immune-related oxidative stress can initiate further increasing levels of oxidative stress that cause organ damage and dysfunction. Targeting oxidative stress in various diseases therapeutically has proven more problematic than first anticipated given the complexities and perversity of both the underlying disease and the immune response. However, growing evidence suggests that the endocannabinoid system, which includes the CB₁ and CB₂ G-protein-coupled receptors and their endogenous lipid ligands, may be an area that is ripe for therapeutic exploitation. In this context, the related nonpsychotropic cannabinoid cannabidiol, which may interact with the endocannabinoid system but has actions that are distinct, offers promise as a prototype for anti-inflammatory drug development. This review discusses recent studies suggesting that cannabidiol may have utility in treating a number of human diseases and disorders now known to involve activation of the immune system and associated oxidative stress, as a contributor to their etiology and progression. These include rheumatoid arthritis, types 1 and 2 diabetes, atherosclerosis, Alzheimer disease, hypertension, the metabolic syndrome, ischemia-reperfusion injury, depression, and neuropathic pain.