Molecular cytology of the respiratory tract and pleura

There is growing evidence that molecular testing is feasible on all types of cytological preparation, which is fortunate as more diagnostic markers and biomarkers for targeted therapies are discovered for use in pulmonary and pleural malignancies. In this article we will discuss the pre-analytic, analytic, and post-analytic (interpretive) considerations for successful implementation of molecular tests for diagnostic and predictive markers in respiratory and pleural cytology. The vast majority of laboratories are familiar with, and have validated their molecular protocols for, formalin-fixed paraffin-embedded surgical specimens, which are not directly applicable to cytology specimens. Thus, rigorous validation must be performed for each type of fixative and cytology preparation before it is implemented in the clinical setting.     

Tumor markers in the detection of recurrent transitional cell carcinoma of the bladder: a brief review

OBJECTIVE: To determine the status of the most promising tumor markers of bladder cancer, including comparison with cytology, technical complexity and utility in patient management. STUDY DESIGN: An extensive literature search was performed, and multiple markers were evaluated. The markers with the greatest potential for use as an adjunct to cytology were reviewed to determine the value of clinical implementation. Markers with a paucity of clinical research and poor results in clinical trials were omitted from review, as were genetic and cytologic prognostic determinants. RESULTS: NMP22, bladder tumor antigen, fibrin/fibrinogen degradation products, telomerase and QUANTICYT image analysis cytometry produced the most favorable and reproducible results. Each test obtained favorable sensitivities in comparison with cytology, especially in the detection of low grade lesions. Many also retrospectively placed patients in high- and low-risk groups based on the test results, allowing increased follow-up time between cystoscopies. However, inability to detect some high grade lesions reduces their utility. CONCLUSION: Continued clinical trials using these and other predictors of bladder cancer will eventually find a test that is suitable, in sensitivity and specificity, for use in urology clinics. Until that time, these tests may be useful in conjunction with cytology to prolong the interval between cystoscopies.     

Diagnostic Utility of the ImmunoCyt/uCyt+ Test in Bladder Cancer

Bladder cancer is a common malignancy in the United States. Although urine cytology is a useful adjunct in both diagnosis and follow-up and is highly sensitive for detecting high-grade tumors, it is limited by decreased sensitivity in detecting low-grade tumors, which constitute the majority of new diagnoses. Additional screening tests with high sensitivity and specificity for urothelial tumors of all grades are indicated to help improve the diagnostic ability of urine cytology as well as to reduce the need for frequent cystoscopies, especially in those with low-risk disease. Several assays have been developed, with the ImmunoCyt/uCyt+ test (DiagnoCure, Inc., Québec, Canada) being especially promising. Recent studies on the applicability and efficacy of ImmunoCyt/uCyt+ testing are reviewed, as are its sensitivity, specificity, and predictive value in the follow-up and screening of urothelial malignancies.     

p53 mutations as tumor markers in fine needle aspirates of palpable breast masses

OBJECTIVE: Mutations in p53 exons 5-8 are found in 40-50% of breast carcinomas. We performed a retrospective analysis of p53 mutations in fluid-based, archival fine needle aspirates (FNAs) of breast masses to determine their potential diagnostic utility as breast tumor cell markers. STUDY DESIGN: Residual, fluid-based, archival FNAs of 27 breast masses were retrospectively evaluated by polymerase chain reaction (PCR), single-strand conformational polymorphism analysis (SSCP) and sequencing for p53 exons 5-8. Results were compared with the morphologic diagnoses and genotyping of available excisional biopsy tissue. RESULTS: Six of the twenty-seven cases were found to have a clonal mutation in p53; all six mutated cases showed carcinoma on subsequent excisional biopsy. Definitive cytologic diagnosis of cancer had been possible in only four of the six cases. Identical mutations were found in the excised carcinomas in the five cases with available tissue. None of the 14 aspirates with benign cytology had detectable mutations in p53. CONCLUSION: p53 Mutational analysis by PCR/SSCP/sequencing deserves to be critically studied as a diagnostic criterion in patients with indeterminate or suspicious cytology. Validation studies should be performed to test p53 mutations as molecular diagnostic markers in breast cytology specimens.     

Critical evaluation of urinary markers for bladder cancer detection and monitoring

Bladder cancer is currently diagnosed using cystoscopy and cytology in patients with suspicious signs and symptoms. These tests are also used to monitor patients with a history of bladder cancer. The recurrence rate for bladder cancer is high, thus necessitating long-term follow-up. Urine cytology has high specificity but low sensitivity for low-grade bladder tumors. Recently, multiple noninvasive urine-based bladder cancer tests have been developed. Although several markers have been approved by the US Food and Drug Administration for bladder cancer surveillance, only a few are approved for detection of bladder cancer in high-risk patients.     

Beyond the suspicious thyroid fine needle aspirate. A review

Fine needle aspiration (FNA) is currently the best diagnostic tool for thyroid nodules. However, the cytologic category of indeterminate or suspicious lesion, which is found in 10-15% of cases, remains a challenge. Since neither clinical presentation nor intraoperative frozen section is often helpful in differentiating these lesions and since surgical procedures for benign and malignant lesions differ, there is a clear need to develop ancillary tests. In this review we identify 12 potential markers of thyroid malignancy that have been examined in thyroid cytologic samples. Although many of these markers hold promise as adjuncts to FNA cytology, multicenter studies have often shown limitations in the predictive value of these assays due to lack of specificity, sensitivity or both. The recent development, however, of tissue microarray techniques to validate promising new markers suggests that improvements in the approach to indeterminate thyroid FNA samples may soon be at hand. This review presents a summary of the issues facing the development of a clinically useful diagnostic test in the differential diagnosis of thyroid nodules.     

"6 markers/5 colors" extended white blood cell differential by flow cytometry.

Electronic white blood cell (WBC) differential by standard cytology (hematology analyzer and visual inspection of blood smears) is limited to five types and identification of abnormal cells is only qualitative, often problematic, poorly reproducible, and labour costing. We present our results on WBC differential by flow cytometry (FCM) with a 6 markers, 5 colors CD36-FITC/CD2-PE+CRTH2-PE/CD19-ECD/CD16-Cy5/CD45-Cy7 combination, on 379 subjects, with detection of 12 different circulating cell types, among them 11 were quantified. Detection of quantitative abnormalities of whole leucocytes, neutrophils, eosinophils, basophils, monocytes, or lymphocytes was comparable by FCM and by standard cytology in terms of sensitivity and specificity. FCM was better than standard cytology in detection and quantification of circulating blast cells or immature granulocytes, with a first lineage orientation in the former case. All cases of lymphocytosis, with lineage assignment, were detected by FCM. FCM identified a group of patients with excess of CD16pos monocytes as those having an inflammatory syndrome. WBC differential by FCM is at least as reliable as by standard cytology. FCM superiority consists in identification and systematic quantification of parameters that cannot be assessed by standard cytology such as lineage orientation of blast cells or lymphocytes, and expression of markers of interest such as CD16 on inflammatory monocytes.     

Multicolor fluorescence in situ hybridization (M-FISH) on cells from urine for the detection of bladder cancer

Bladder cancer is the fifth most common cancer in adults. Because of the high recurrence rate (up to 70%) new tumor markers for urine are necessary for monitoring patients. In this study, we investigated the value of M-FISH on cells from urine for the detection of bladder cancer. Urine samples from 141 patients suspicious of bladder cancer were analyzed in this study. Cells were isolated from urine before surgical therapy. For FISH analysis, a commercial kit (UroVysion) containing hybridization probes for chromosomes 3, 7, 9p21 and 17, was used. Twenty-five cells were analyzed in each case by two observers. A FISH result was obtained in 121 cases. Overall, sensitivity was 60% and specificity reached 82.6%. Sensitivity and specificity by cytology were 24.1% and 90.5%, respectively. Analyzing results concerning T-category, sensitivity of FISH and cytology was 36.1% and 15% in pTa, 65.2 and 25.7% in pT1, 100% and 66.7% in pT2-3 tumors, respectively. Concerning tumor grade, similar results were obtained: sensitivity was 37% and 14% in G1, 65.4% and 40% in G2, 91.7% and 50% in G3 tumors, for FISH and cytology, respectively. In conclusion, FISH on cells from urine has been shown in all studies to be highly sensitive and specific for detection of bladder cancer. Sensitivity of FISH is higher than conventional cytology and can be used in routine diagnosis additionally to conventional cytology especially in doubtful or negative cases. FISH can detect recurrence earlier than other methods like cytology, cystoscopy or biopsy histological examination.     

Tumor markers, liver function tests and symptoms in 115 patients with isolated colorectal liver metastases

Development of the hybridoma technique has made the identification of several new tumor antigens possible. Although it was hoped that they would be more tumor-specific, none of these markers are found exclusively in tumor or in serum of tumor patients. Compared with carcinoembryionic antigen (CEA) and liver function tests, the roles of these markers (CA 19-9, CA 125, CA 15-3) were prospectively evaluated in 115 patients with colorectal liver metastases. Patients were classified according to tumor volume (T1 less than 25%, T2 25-75%, T3 greater than 75%), and the extension of infiltration (solitary/multiple/diffuse; unilateral, bilateral). Patients with benign liver or biliary disease served as a control group (n = 63). Overall sensitivity was 87% for *1, 50% for *2 and 38% for *3, with a significant correlation with tumor size. CEA serum levels were elevated in 88% of all patients. CA 19-9 was less sensitive: positive in 59%. Because of some complementary elevations, the combined use of CEA, CA 19-9 and CA 125 raised sensitivity to 94%. CA 19-9 and LDH could be useful for confirmation because of their higher specificity; however, the specificity of CEA rose to 93% on using a cut-off of 10 ng/ml instead of 3 ng/ml. The results indicate that CEA and CA 19-9 as well as liver function tests are helpful for preoperative staging in conjunction with imaging procedures before liver resection or regional chemotherapy.     

Dysregulation of MicroRNAs in Colonic Field Carcinogenesis: Implications for Screening

Colorectal cancer (CRC) screening tests often have a trade-off between efficacy and patient acceptability/cost. Fecal tests (occult blood, methylation) engender excellent patient compliance but lack requisite performance underscoring the need for better population screening tests. We assessed the utility of microRNAs (miRNAs) as markers of field carcinogenesis and their potential role for CRC screening using the azoxymethane (AOM)-treated rat model. We found that 63 miRNAs were upregulated and miR-122, miR-296-5p and miR-503# were downregulated in the uninvolved colonic mucosa of AOM rats. We monitored the expression of selected miRNAs in colonic biopsies of AOM rats at 16 weeks and correlated it with tumor development. We noted that the tumor bearing rats had significantly greater miRNA modulation compared to those without tumors. The miRNAs showed good diagnostic performance with an area under the receiver operator curve (AUROC) of >0.7. We also noted that the miRNA induction in the colonic mucosa was mirrorred in the mucus layer fecal colonocytes isolated from AOM rat stool and the degree of miRNA induction was greater in the tumor bearing rats compared to those without tumors. Lastly, we also noted significant miRNA modulation in the Pirc rats- the genetic model of colon carcinogenesis, both in the uninvolved colonic mucosa and the fecal colonocytes. We thus demonstrate that miRNAs are excellent markers of field carcinogenesis and could accurately predict future neoplasia. Based on our results, we propose an accurate, inexpensive, non-invasive miRNA test for CRC risk stratification based on rectal brushings or from abraded fecal colonocytes.     

Cervical cytology screening and government policy.

OBJECTIVE--To assess the coverage of cervical cytology screening in one general practice surgery according to the criteria in the new Scottish general practitioner contract and to explore the difficulties of defining performance in such screening. DESIGN--Review of annual analysis of uptake of screening during 1984-8. SETTING--Suburban general practice surgery in Glasgow serving 3000 patients. PATIENTS--All women aged 35-64 registered in 1984 increasing in 1988 to all women aged 20-64. MAIN OUTCOME MEASURE--Assessment of uptake of smear tests and reasons for smear not being taken and of the effect of these outcomes on the new general practitioner contract. RESULTS--The numbers and percentages of women having a smear test in the previous five and three years were recorded, and the reasons why a smear was not taken were defined in the remainder (hysterectomy, test not offered, risk not known, test declined, patient moved away, and patient unaccounted for). In 1988, 85% (608/719) of the women aged 30-64 and 80% (693/870) of those aged 20-64 had had a smear test in the previous five years. An appropriate or irreducible reason for the lack of a smear test was defined in all the others. CONCLUSIONS--The population studied contained a substantial number of women in whom cervical smear was unnecessary, inappropriate, or refused. These factors and the likely demographic variation in the uptake of smear tests have important implications for the setting and achieving of the government''s targets for cervical cytology screening.     

Genetic Defects as Tumor Markers

Carcinogenesis is long-term multistep accumulation of defects of genes responsible for cell division, DNA repair, and apoptosis. The functions of these genes are known both for norm and for pathologies caused by their damage and resulting in asocial cell behavior. Owing to the recent progress in studying the mechanisms of carcinogenesis, some genetic defects may be considered from the applied point of view (as tumor markers rather than as pathogenetic factors) and employed in diagnostics. Thus detection of mutant alleles in biological fluids (e.g., beyond the tumor) suggests higher risk of carcinogenesis. Genetic defects are a new class of tumor markers and have a substantial diagnostic potential. In contrast to known protein markers (-fetoprotein, etc.) used in clinical practice, DNA markers are oncospecific (as these are in direct cause-and-effect relationships with carcinogenesis) and universal (as there is not a single tumor cell without a genetic defect). Analysis of DNA markers may be employed not only in diagnostics or tumor growth monitoring (assessment of treatment efficiency, early detection of recurrence or metastasis), but also (prospectively) in screening (tumor detection at the presymptomatic stage, identification of high-risk groups). Theoretical grounds, prospects, problems, and methods of this new field are considered.     

Which women default from follow‐up cervical cytology tests? A cohort study within the TOMBOLA trial

L. Sharp, S. Cotton, A. Thornton, N. Gray, D. Whynes, L. Smart, N. Waugh, I. Duncan, M. Cruickshank and J. Little, on behalf of the TOMBOLA Group Which women default from follow‐up cervical cytology tests? A cohort study within the TOMBOLA trial Objective: To identify factors associated with default from follow‐up cervical cytology tests. Methods: A cohort study was conducted involving 2166 women, aged 20–59, with recent low‐grade cervical cytology taken within the NHS Cervical Screening Programmes in Scotland and England, and managed by 6‐monthly cytology in primary care. For the first (6‐month) and second (12‐month) surveillance cytology tests separately, women were categorized as ‘on‐time attendees’ (attended ≤6 months of test being due), ‘late attendees’ (attended greater than 6 months after test was due) or ‘non‐attendees’ (failed to attend). Multivariate odds ratios (ORs) were computed for factors associated with late and non‐attendance. Results: For the first surveillance test, risk of non‐attendance was significantly higher in younger women, those without post‐secondary education, and non‐users of prescribed contraception. Factors significantly associated with late attendance for the first test were the same as for non‐attendance, plus current smoking and having children. The most important predictor of non‐attendance for the second surveillance test was late attendance for the first test (OR = 9.65; 95% CI, 6.60–16.62). Non‐attendance for the second test was also significantly higher among women who were younger, smokers and had negative cytology on the first surveillance test. Late attendance for the second surveillance test was higher in women who were younger, smokers, had children and attended late for the first test. Conclusions: Women at highest risk of default from follow‐up cytology tend to be young, smoke, lack post‐secondary education, and have defaulted from a previous surveillance appointment. Tackling default will require development of targeted strategies to encourage attendance and research to better understand the reasons underpinning default.     

Personal view. Is it reality or an illusion that liquid‐based cytology is better than conventional cervical smears?

Liquid-based cytology (LBC) has been heralded as the way forward for cervical screening, and as the answer to many of its problems. It is already used as a sole method of cell preparation in many private clinics in the UK. It is being used for colposcopy smears in many NHS clinics and is now being piloted for primary screening in three screening centres in England, as well as one in Scotland and one in Wales. LBC has been welcomed as a new technology because it deals with the problem of specimen adequacy at source, removing responsibility for slide preparation and fixation from the clinician or nurse. It provides uniformly well-fixed preparations that are free of inflammatory exudate and blood, and seem easier to screen than conventional smears. There are many articles in the world literature suggesting that LBC is more accurate than conventional screening, and it is thought likely to reduce the number of false negative tests. The main reasons for piloting LBC in the NHS Cervical Screening Programme (NHSCSP) lie in its potential for reducing screening times and for reducing the numbers of repeats for inadequate tests. LBC is expensive in terms of equipment, capital costs, maintenance, consumables, training, technical preparation time, transportation and disposal of liquid media. Its costs could be justified if they were offset by the money saved from reduced screening time and repeat tests, but only if its accuracy in terms of sensitivity and specificity were proven to be equal to or better than conventional cytology. Although that is generally held to be true by the public and medical profession alike, there is very little hard evidence to support it.     

Interwoven dendritic processes of follicular dendritic cell sarcoma demonstrated on ultrafast papanicolaou-stained smears: a case report

BACKGROUND: Follicular dendritic cell (FDC) tumor is a rare tumor derived from accessory cells in the lymphoid follicles. FDC tumors are typically diagnosed on histology based on immunoreactivity to at least 1 of the FDC markers (CD21, CD23 or CD35) or based on the characteristic ultrastructural feature of long, interwoven, cytoplasmic, dendritic processes connected by desmosomes. CASE: We observed novel cytologic features of FDC sarcoma in a liver fine needle aspirate of a 46-year-old man status post surgery and chemotherapy for FDC sarcoma, originating in the gastrointestinal tract with metastases to the liver, pancreas and spleen. In the Diff-Quik- and Papanicolaou-stained smears, the tumor cells presented in syncytial fragments as well as single cells, as previously reported in the cytologic literature. However, the single cells were interconnected to neighboring single cells via long, thin, threadlike cytoplasmic processes in ultrafast Papanicolaou (UFP)-stained smears. The tumor cells possessed multipolar cytoplasmic processes rather than unipolar ones, as previously reported. CONCLUSION: The ultrastructural features of a web of interwoven, dendritic, cytoplasmic processes of FDC tumor was demonstrated for the first time on cytology. Observation of this feature may allow the diagnosis to be made on cytology prior to histology, immunohistochemistry or electron microscopy.     

Genetic defects as markers of tumor development

Carcinogenesis is long-term multistep accumulation of defects of genes responsible for cell division, DNA repair, and apoptosis. The functions of these genes are known both for norm and for pathologies caused by their damage and resulting in "asocial" cell behavior. Owing to the recent progress in studying the mechanisms of carcinogenesis, some genetic defects may be considered from the applied point of view (as tumor markers rather than as pathogenetic factors) and employed in diagnostics. Thus detection of mutant alleles in biological fluids (e.g., beyond the tumor) suggests higher risk of carcinogenesis. Genetic defects are a new class of tumor markers and have a substantial diagnostic potential. In contrast to known protein markers (alpha-fetoprotein, etc.) used in clinical practice, DNA markers are oncospecific (as these are in direct cause-and-effect relationships with carcinogenesis) and universal (as there is not a single tumor cell without a genetic defect). Analysis of DNA markers may be employed not only in diagnostics or tumor growth monitoring (assessment of treatment efficiency, early detection of recurrence or metastasis), but also (prospectively) in screening (tumor detection at the presymptomatic stage, identification of high-risk groups). Theoretical grounds, prospects, problems, and methods of this new field are considered.     

基因组学与表观遗传学在肿瘤标志物研究中的应用

肿瘤标志物是一类能反映肿瘤存在与生长状态的生化物质,它们在肿瘤的早期筛查、辅助诊断、预后判断、疗效评价、复发和转移监测中具有重要意义. 随着细胞与分子生物学领域的发展,人类基因组计划大量研究成果的涌现,人们对肿瘤的发生和发展机理有了越来越清楚的认识,肿瘤标志物的研究也得到了极大的拓展与更新,特别是基因组学和表观遗传学的进展大大促进了新型肿瘤标志物的发现. 文章就肿瘤标志物发展历程,常用肿瘤标志物的种类和临床应用进行介绍,分析了现有肿瘤标志物的局限性,重点阐述了新型肿瘤标志物,如多种表观遗传学标志物及循环核酸的研究及应用. 同时,对肿瘤标志物的研究前景提出了一些观点.     

基于Solexa高通量测序的香菇C_(91-3)功能基因的挖掘和开发

香菇是全球人工种植最为普遍的食用菌,近年来,国内外的研究者对香菇进行了深入的研究,发现香菇在预防、治疗恶性肿瘤方面都具有重要的药用价值。尤其是香菇多糖制剂现已正式作为辅助化疗药物应用于临床。但是对香菇中蛋白质生物学活性的研究以及从蛋白质水平去发掘其抗肿瘤活性的功能基因却鲜有报道。本文拟就香菇的基因组、转录组及功能基因挖掘和开发现状进行简要论述。     

Vaginal vault cytology tests: analysis of a decade of data from a UK tertiary centre

H. Stokes‐Lampard, S. Wilson, C. Waddell and L. Bentley Vaginal vault cytology tests: analysis of a decade of data from a UK tertiary centre Objectives: To examine temporal trends in the use of vault cytology tests in primary and secondary care and the demographics of those women tested. Methods: Retrospective analysis of routinely collected data concerning women who had a vault cytology test processed during a 10‐year period (1 April 1995 to 31 March 2005) at Birmingham Women’s NHS Foundation Trust. Results: A total of 8457 vault cytology tests from 3164 women (range 1–17 tests, median = 2) were processed, representing approximately 2% of the cervical cytology workload of the Department of Cytopathology at Birmingham Women's Hospital. There was a significant reduction in annual numbers processed (Pearson correlation ?0.958, P < 0.001). Significant abnormalities (mild dyskaryosis or worse) were detected in 4.5%, with malignancy being detected in <0.1%. The unsatisfactory cytology test rate was 10.7% overall. There was a reduction in the numbers of vault cytology tests coming from the community, hospital outpatient clinics and operating theatres over time (χ² for linear trend = 139.53, 9 d.f., P < 0.0001). Tests originating from community settings had the lowest disease detection rates: no malignancies and only two severe abnormalities were detected from almost 4000 primary care samples; abnormal results represented 2.8% (n = 113), of which the majority (n = 73) were borderline results. All cancers (n = 8) were detected in samples taken in gynaecology and colposcopy clinics. Conclusions: Vault cytology test usage appears to be reducing, particularly from outpatient clinics and primary care. Community detection rates are very low. Further research is required to establish the true costs and benefits of vaginal vault cytology.     

Accuracy of the analysis of multiple small fragments of glial tumors obtained by stereotactic biopsy.

To examine the reliability of the diagnoses reached on multiple small fragments of cerebral glial tumors obtained via stereotactic biopsy, samples obtained from 100 consecutive glial tumors (during real or simulated biopsy) were studied by cytology and histology. In comparison to the definitive diagnosis made on the whole tumor, a correct positive diagnosis on the biopsy sample was made by histology in 96% of cases and by cytology in 93% of the cases (with 96% correct results when combining both methods). A correct identification of the tumor type and grade was achieved by histology in 82% of cases and by cytology in 80% of the cases (with 85% correct results when combining both methods). The limits of stereotactic biopsy are related to the difficulty of identifying all of the typical tumor features on tiny tissue fragments of a pleomorphic neoplasm, such as a glioma. This study demonstrates that better results may be obtained by using both cytology and histology to study multiple stereotactic biopsy samples from glial tumors.