Intraperitoneal-Free Cancer Cells Represent a Major Prognostic Factor in Colorectal Peritoneal Carcinomatosis.

BACKGROUND: Intraperitoneal-free cancer cells are considered as an important prognostic tool in gastric and ovarian cancer. However, their significance in colorectal cancer remains more controversial. OBJECTIVE: This study aimed to determine the role of intraperitoneal-free cancer cells as a prognostic tool in the outcome in colorectal peritoneal carcinomatosis treated with curative intent by complete cytoreductive surgery. DESIGN: This study is an analysis of a prospectively maintained database. PATIENTS: Between 1991 and 2012, all patients treated in a single institution for colorectal peritoneal carcinomatosis by complete cytoreductive surgery with peritoneal cytology available were evaluated. Peritoneal cytology was stained in the conventional way (May Grumwald Giemsa). RESULTS: Among a population of 162 patients treated for colorectal peritoneal carcinomatosis by complete cytoreductive surgery, 38 presented positive intraperitoneal-free cancer cells (23.5%). Systemic chemotherapy was administered to 135 patients (85%) during the preoperative course. Median follow-up was 34.5 months. Median overall survival was 19 and 44 months for positive and negative intraperitoneal-free cancer cells (p = 0.018). In multivariate analysis, Peritoneal Carcinomatosis Index and positive intraperitoneal-free cancer cells were significant prognostic factors of overall survival (HR, 2.3 (1.18-4.52), p = 0.014; HR, 1.9 (1.08-3.38), p = 0.027). LIMITATIONS: Retrospective analysis and the long period were limitations of study. CONCLUSION: Along with the Peritoneal Carcinomatosis Index, intraperitoneal-free cancer cells are a strong prognostic factor for patients treated with curative intent for colorectal peritoneal carcinomatosis by complete cytoreductive surgery. The presence of intraperitoneal-free cancer cells should lead to the consideration of different treatment strategies such as extensive intraperitoneal lavage, targeted intraperitoneal therapies, or repeated intraperitoneal chemotherapy.

Guidelines for May-Grünwald-Giemsa staining in haematology and non-gynaecological cytopathology: recommendations of the French Society of Clinical Cytology (SFCC) and of the French Association for Quality Assurance in Anatomic and Cytologic Pathology (AFAQAP).

OBJECTIVE: Since the guidelines of the International Committee for Standardisation in Haematology (ICSH) in 1984 and those of the European Committee for External Quality Assessment Programmes in Laboratory Medicine (EQALM) in 2004, no leading organisation has published technical recommendations for the preparation of air-dried cytological specimens using May-Grünwald-Giemsa (MGG) staining. DATA SOURCES: Literature data were retrieved using reference books, baseline-published studies, articles extracted from PubMed/Medline and Google Scholar, and online-available industry datasheets. RATIONALE: The present review addresses all pre-analytical issues concerning the use of Romanowsky's stains (including MGG) in haematology and non-gynaecological cytopathology. It aims at serving as actualised, best practice recommendations for the proper handling of air-dried cytological specimens. It, therefore, appears complementary to the staining criteria of the non-gynaecological diagnostic cytology handbook edited by the United Kingdom National External Quality Assessment Service (UK-NEQAS) in February 2015.

Genotyping of high-risk human papillomaviruses in p16/Ki-67-positive urothelial carcinoma cells: even a worm will turn.

OBJECTIVE: Co-expression of p16INK4a protein and Ki-67 (p16/Ki-67) is noted in almost all high-grade urothelial lesions. However, the aetiological role or, conversely, the absence of causative effect of high-risk human papillomaviruses (hr-HPVs) has not been documented. The purpose of this study is to evaluate HPV DNA in p16/Ki-67-positive, high-grade urothelial tumour cells. METHODS: Fifty-seven urine samples collected from 50 patients, including 55 histologically proven high-grade proliferations and two cases with clinical evidence of malignancy, were analysed for p16/Ki-67. Immunolabelling was performed in destained Papanicolaou-stained slides after ThinPrep(®) processing. HPV genotyping was performed by polymerase chain reaction (PCR) using a DNA microarray for 35 HPV types. Confirmation of the presence (or absence) of HPV in tissue samples was verified using a reasoned approach combining PCR and in situ hybridization (ISH) for hr-HPVs. RESULTS: Co-expression of p16/Ki-67 was noted in 43 of 57 (75.4%) cases. In these, hr-HPVs 16, 31 and 70, and low risk HPV 84, were detected in the urine in four patients (8%). Upregulation of p16INK4a protein was confirmed on bladder biopsy or transurethral resection specimens, but PCR and ISH for hr-HPVs were both negative on the tissue sections. CONCLUSION: Our results show a low prevalence of HPV infection in the urinary tract of patients with p16/Ki-67-positive urothelial malignancy. The study confirms that the deregulated cell cycle, as demonstrated by p16/Ki-67 dual labelling, is independent of the oncogenic action of hr-HPVs in high-grade urothelial proliferations.

Diagnostic terminology for urinary cytology reports including the new subcategories 'atypical urothelial cells of undetermined significance' (AUC-US) and 'cannot exclude high grade' (AUC-H).

OBJECTIVE: We studied whether atypical, non-superficial urothelial cells (AUC) could be separated into new subcategories including AUC 'of undetermined significance' (AUC-US) and 'cannot exclude high grade'' (AUC-H) in order to help to standardize urine cytopathology reports, as it is widely accepted in the Bethesda system for gynaecological cytopathology. METHODS: We investigated whether AUC-US and AUC-H, defined by distinctive cytological criteria, might be separated with statistical significance according to actual diagnosis and follow-up data. A series of 534 cyto-histological comparisons taken in 139 patients, including 221 AUC at various steps of their clinical history was studied. There were 513 (96.1%) postcystoscopy and 469 (87.8%) ThinPrep® liquid-based specimens (95.9% and 89.1% of AUC cases, respectively). Patients viewed between 1999 and 2011 had histological control in a 0- to 6-months delay and were followed-up during an additional 5.9 ± 9.2 (0- to 56-) months period. RESULTS: The 221 AUC represented 0.8-2% of the specimens viewed during the study period. Among AUC-H cases, 70 out of 185 (37.8%) matched with high-grade lesions, compared with 3 of 38 (8.3%) of AUC-US cases (P = 0.0003). Conservatively treated patients with AUC-H more frequently developed high-grade lesions than those with AUC-US (54.1% versus 16.7%, P = 0.0007) with a 17.6-months mean delay. Nuclear hyperchromasia, a nuclear to cytoplasm (N/C) ratio > 0.7 and the combination of both were the more informative diagnostic criteria, all with P < 0.01. CONCLUSION: We conclude that the new subcategories could help to standardize urine cytopathology reports and contribute to the patient's management, provided it is validated by multicentric studies.

Lack of prognostic significance of conventional peritoneal cytology in colorectal and gastric cancers: results of EVOCAPE 2 multicentre prospective study.

AIM: In digestive cancers, the prognostic significance of intraperitoneal free cancer cells remains unclear (IPCC). The main objective of this study was to assess the prognostic significance of IPCC in colorectal and gastric adenocarcinoma. The secondary objectives were to evaluate the predictive significance of IPCC for the development of peritoneal carcinomatosis (PC) and to evaluate the prevalence of synchronous PC and IPCC. METHODS: This was a prospective multicentre study. All patients undergoing surgery for a digestive tract cancer had peritoneal cytology taken. Patients with gastric and colorectal cancer with no residual tumour after surgery and no evidence of PC were followed-up for 2 years. The primary end point was overall survival. RESULTS: Between 2002 and 2007, 1364 patients were enrolled and 956 were followed-up over 2 years. Prevalence of IPCC was 5.7% in colon cancer, 0.6% in rectal cancer and 19.5% in gastric cancer. The overall 2-year survival rate for patients with IPCC was 34.7% versus 86.8% for patients with negative cytology (p<0.0001). By multivariate analysis, IPCC was not an independent prognostic factor. No relationship between cytology and recurrence was found. CONCLUSION: The presence of IPCC was not an independent prognostic and didn't add any additional prognostic information to the usual prognostic factors related to the tumour (pTNM and differentiation). Moreover the presence of IPCC detected with this method didn't appear to predict development of PC. Peritoneal cytology using conventional staining doesn't seem to be a useful tool for the staging of colorectal and gastric cancers.

p16(INK4a) /Ki-67 dual labelling as a marker for the presence of high-grade cancer cells or disease progression in urinary cytopathology.

OBJECTIVE: Overexpression of p16(INK4a) independent of the presence of E6-E7 oncoproteins of high-risk papillomaviruses has been identified in bladder carcinoma in situ lesions with or without concurrent papillary or invasive high-grade (HG) urothelial carcinoma. As p16(INK4a) and Ki-67 co-expression clearly indicates deregulation of the cell cycle, the aim of this study was to investigate the frequency of p16(INK4a) /Ki-67 dual labelling in urinary cytology samples. METHODS: Immunolabelling was performed in demounted, destained Papanicolaou slides after ThinPrep(®) processing. A total of 84 urinary cytology samples (18 negative, 10 low grade, 19 atypical urothelial cells and 37 high grade) were analysed for p16(INK4a) /Ki-67 co-expression. We assessed underlying urothelial malignancy with cystoscopy, histopathology and follow-up data in every case. RESULTS: Compared with raw histopathological results, p16 (INK4a) /Ki-67 dual labelling was observed in 48 out of 55 (87.3%) HG lesions and in 11 out of 29 (37.9%) negative, papillary urothelial neoplasia of low malignant potential or low-grade carcinomas (P = 0.05). All cases with high-grade/malignant cytology were dual labelled. Sixteen out of 17 (94.1%) carcinoma in situ cases and eight out of 14 (57.1%) cases with atypical urothelial cells matching with HG lesions were dual labelled. Extended follow-up allowed three cases of progression to be diagnosed in dual-labelled cases with negative/low-grade cytology results after a 9- to 11-months delay. CONCLUSIONS: The data show that p16(INK4a) /Ki-67 co-expression allows most HG cancer cells to be detected initially and in the follow-up period. Additional studies are needed in order to determine whether dual labelling can be used as a triage tool for atypical urothelial cells in the urine.

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.

Intraperitoneal free cancer cells in non-gynaecological adenocarcinomas: a reproducibility study.

OBJECTIVE: In recent years, therapeutic approaches including cytoreductive surgery followed by intraperitoneal chemotherapy have proven effective in peritoneal carcinomatosis of colorectal origin. If cytology is to be used to include patients in aggressive treatment regimens, it is necessary to evaluate its performance, particularly in terms of specificity. The aim of this study was to assess interobserver agreement for the detection of intraperitoneal free cancer cells (IFCCs) in patients with non-gynaecological adenocarcinomas. METHODS: Over a 5-year period, 1223 patients were recruited in 19 French surgery departments. Peritoneal samples were examined in 14 dispersed pathology laboratories. Giemsa-stained slides were sent to a control reader blind to the previous diagnosis. Discordant cases, concordant positive results and a random selection of negative concordant cases were reviewed by a panel of seven cytopathologists. The 'final diagnosis' was that of the consensus meetings but took into account locally-processed slides. RESULTS: Gathering dubious cases with negative results, a 95.6% concordance was achieved between local readers and the control reader. IFCCs were ascertained by the panel in 85 cases (7.0%). Eight of 873 colorectal cancers cases viewed locally were falsely positive (0.9%). Radiotherapy and neoadjuvant therapy had no impact on the false-positive rate as assessed by final validation by the panel (P > 0.05). Samples initially considered as dubious were reclassified as negative by the panel in 24 of 25 cases (96.0%). CONCLUSIONS: The panel consensus allowed reclassification of most dubious/equivocal peritoneal cytology cases, whereas clearcut distinction between benign and malignant cases was correctly achieved in almost all cases.

p53 immunodetection of liquid-based processed urinary samples helps to identify bladder tumours with a higher risk of progression.

p53 could help identify bladder tumour cases with a risk of progression from superficial to invasive disease. Semiautomatic, liquid-based cytology (LBC) techniques offer an opportunity to standardise molecular techniques. The aim of our study was to investigate whether LBC could improve p53 immunolabelling, and to assess whether urinary p53 could have a prognostic value. Immunoreactivity for p53 was studied in 198 urine samples after treatment with the Cytyc Thinprep processor. After antigen retrieval, cells were labelled with a monoclonal antibody that recognises both wild-type and mutant form of the p53 protein (Clone DO-7, Dako), 1/1000. Positivity for p53 was assessed in 17.2% of the cases. High-grade (G3) tumours were positive in 74.1% of the cases. Comparatively, low-grade (G1-2) urothelial carcinomas were positive in 23.5% of the cases. During a median follow-up period of 26 months, recurrence was observed in 52.9% of the cases with p53 overexpression, and in only 10.9% of negative cases (P < 0.001). The progression rate was 35.3% of p53-positive cases vs 5.5% of p53-negative cases (P < 0.001). Progression-free survival was significantly shorter in patients with p53 accumulation (P = 0.007). In a multivariate analysis stratified on grade and stage, p53 was an independent predictor of overall survival (P = 0.042). The results show that using Thinprep LBC, p53 immunolabelling of voided urothelial cells allows most high-grade tumours to be detected and may help identify cases with a higher risk of recurrence and progression.

Cost efficiency analysis of modern cytocentrifugation methods versus liquid based (Cytyc Thinprep) processing of urinary samples.

BACKGROUND/AIMS: Liquid based cytology (LBC) was developed as a replacement for cytocentrifugation in the treatment of cell suspensions. Because accurate data comparing the quality and total cost of modern cytocentrifugation methods versus LBC in non-gynaecological samples are not available, this study was designed to investigate these issues. METHODS: The study comprised 224 urine samples treated with the Thermo Shandon Cytospin 4 using reusable TPX chambers, disposable Cytofunnels for samples up to 0.5 ml, and disposable Megafunnels for samples up to 6 ml. Each method was compared with the Cytyc Thinprep processing of a paired sample. Quality was assessed by scoring cellularity, fixation, red blood cells, leucocytes, abnormalities of urothelial cells, and suitability for molecular studies. Wage costs, investment, and consumables allowed a "total cost" to be calculated on the basis of 200 specimens/month. Total cost and quality combined were used to calculate an index of total quality (ITQ). RESULTS: Cytocentrifugation with disposable chambers resulted in a global quality superior to that of Cytyc Thinprep LBC. Preparation and screening times were 2.25 and 1.33-2 times greater when using LBC compared with cytocentrifugation. The total cost each month reached 1960.23 $ to 2833.43 $ for cytocentrifugation methods and 5464.95 $ for Cytyc Thinprep LBC (92.8-178.8% increased cost). ITQ of cytocentrifugation with disposable chambers surpassed that of Cytyc Thinprep LBC (37.25/32.08 and 9.98, respectively). CONCLUSION: Cytyc Thinprep LBC and cytocentrifugation are both appropriate methods for cytology based molecular studies, but cytocentrifugation remains the quality standard for current treatment of urinary samples because of its lower cost.

Flow and image cytometric DNA measurements in fine needle aspiration samples of prostatic neoplasms.

OBJECTIVE: To compare the DNA content measured by flow cytometry (FCM) and image analysis (IA) from prostatic fine needle aspiration (FNA) samples. STUDY DESIGN: A total of 48 samples were studied. FCM was performed on propidium iodide-stained nuclei according to the Vindelov method, and image analysis was performed on Feulgen-stained slides. Positive FNA results were grade (1-3) and compared with Gleason grades. RESULTS: Aneuploidy was closely related to positive FNA results (P < .01). DNA histograms were found to be aneuploid in all grade 3 carcinomas (n = 13) by IA and in 11 cases (84.6%) by FCM. Grade 2 carcinomas (n = 9) were found to be aneuploid with both methods. In grade 1 carcinomas (n = 10), 2 cases exhibited IA aneuploid profiles, whereas all FCM cases were diploid. Aneuploid profiles were more often associated with high Gleason scores than were diploid ones (P < .01). Among the 16 patients with negative FNA results, two cases had tetraploid DNA profiles related to contaminating seminal vesicle cells. The difference in DNA measurements reached 10.4% but was not statistically significant. CONCLUSION: These findings show that the two methods, as applied to prostatic FNA samples, give comparable results and that seminal vesicle cells may be responsible for false tetraploid profiles.

Bracken fern-induced bladder tumors in guinea pigs. A model for human neoplasia.

We have induced tumors by feeding guinea pigs with a diet containing 25 or 30% dried bracken fern for 100 or 150 days. A high incidence of bladder tumors was obtained. All but one animal had preneoplastic or neoplastic lesions after 4 months; after one year, 24 or 25 exposed animals had carcinoma. Bladder tumors obtained were essentially pure transitional cell carcinomas, although 4 cases (7% of the exposed animals and 10% of the 39 transitional cell carcinoma observed) showed areas of focal squamous metaplasia. Immunohistological detection of cytokeratins 10, 13, and 18 confirmed the transitional nature of these tumors. Tumor development can be followed by ultrasonography and cytology. Bladder tumors arose through several steps. Dysplasia and preneoplastic hyperplasia were seen after 4 months and papillary carcinomas appeared after 6 months, whereas muscle-invasive carcinomas required 1 year. Thus this model reproduces the full spectrum of preneoplastic and neoplastic bladder lesions observed in humans. Interestingly, when tumors were induced in older guinea pigs, none of them progressed to a muscle-invasive stage. This phenomenon should provide the opportunity to study the molecular mechanisms associated with these two different growth patterns, a major issue in understanding human bladder tumor progression.

Clinical value of fine-needle aspiration cytology and biopsy in the evaluation of male infertility. A comparative study of 48 infertile patients.

OBJECTIVE: To evaluate whether fine-needle aspiration cytology of the testis can be considered as a diagnostic parameter in the evaluation of male infertility. PATIENTS AND METHODS: We studied 30 oligospermic and 18 azoospermic patients using 63 fine-needle aspiration samples and 57 biopsy samples obtained surgically (10 cases) or with a spring-loaded biopsy device (47 cases). Cytologic evaluation of spermatogenesis was performed by studying longitudinal segments of seminiferous tubules and cytocentrifuged dissociated cells. RESULTS: Comparison between fine-needle aspiration and the biopsy methods gave concordant results in 72.2% of cases. Discordant findings were recorded in 10 cases (27.8%). In eight cases, significant maturation into spermatozoa was recognized in samples obtained by fine-needle aspiration only, whereas moderate to severe hypospermatogenesis or germ cell aplasia were demonstrated in samples obtained by the spring-loaded biopsy device or by open surgical biopsy. Germ cell aplasia was recognized in samples obtained by both methods in 75.0% of cases. Insufficient specimens were obtained by fine-needle aspiration and the spring-loaded biopsy device in 15.9% and 12.3% of cases, respectively, whereas all surgical biopsy specimens were of good quality. Four bleeding episodes and one case of epididymitis were observed after use of the spring-loaded biopsy device, but no complication was related to either surgical biopsy or fine-needle aspiration. CONCLUSION: The findings show that fine-needle aspiration cytology could represent a more reliable means of identifying significant numbers of the most mature germ cells.

Prospective study of combined use of bronchial aspirates and biopsy specimens in diagnosis and typing of centrally located lung tumours.

OBJECTIVE: To determine the diagnostic accuracy of examining bronchial secretions in pulmonary cytopathology and whether cytology and histopathology can complement each other in routine practice among lung specialists. DESIGN: A prospective study comparing 1225 cytological and biopsy results, conducted during 1987-93. Tumours were confirmed by histopathology, imaging techniques, or clinical outcome and imaging techniques combined. SETTING: 11 lung or internal medicine units, France. SUBJECTS: 1128 patients (874 men; 254 women) aged 65.3 (SD 13.7) years who underwent fibreoptic bronchoscopy for various pulmonary symptoms. RESULTS: Exact concordance between cytological and biopsy results was obtained in 1036/1187 (87.3%) satisfactory specimens. In all 574 lung tumours were diagnosed. One case (0.08%) was a false positive cytological diagnosis in a patient with tuberculosis. Patients with lung cancer were more likely to have positive cytological results than positive biopsy results (P < 0.001). Agreement in tumour typing was observed in 375/424 (88.4%) cases, when non-small cell carcinomas, small cell carcinomas and undifferentiated carcinomas were separated. In the 11 patients with squamous cell carcinomas in situ, eight (72.7%) of the carcinomas were diagnosed cytologically as squamous cell. Unsatisfactory material was obtained in only 20 (1.6%) and 19 (1.6%) cases by cytology and biopsy respectively. Examinations had to be repeated in 86 (7.6%) patients. CONCLUSIONS: Examination of bronchial secretions complements histopathology in both diagnosing and typing lung tumours and could be performed more systematically in patients undergoing fibreoptic bronchoscopy.

[Bronchial aspiration in the typing of bronchopulmonary cancers: apropos of 150 cytobiopsy correlations]. / L'aspiration bronchique dans le typage des cancers bronchopulmonaires: à propos de 150 corrélations cytobiopsiques.

We analysed 150 cases of primary lung cancers investigated by bronchial aspirate and biopsy methods with clinical, radiologic and bronchoscopic findings. Among the 150 cases studied, three were characterized by mixed tumor cell components, thus allowing 153 cyto-histological comparisons. The cytologic and histopathologic typing agreed strictly in 102 cases of 153 (66.7%) and was considered as correct in 40 cases (26.1%). Only cases with divergent evaluation between small-cell carcinoma and non small-cell carcinoma were considered as discordant: such misclassification occurred in 11 specimens (7.2%), including two cases with mixed patterns. The cytologic typing was in agreement with the final diagnosis in all epidermoid carcinomas, adenocarcinomas, large-cell carcinomas and poorly differentiated carcinomas. In small-cell carcinomas, cytology was in agreement with histopathology in 20 of 26 cases (77%), and could only indicate undifferentiated features in four cases (15.4%). The analysis of bronchial aspirate specimens gave reliable typing in 92% of cases, and indicated a better tumor cell differentiation than histopathology in 6.5% of cases. The results obtained show that aggressive treatments can be reliably proposed on the basis of cytologic findings, even without tissue corroboration. This proposal is particularly helpful in cases where biopsy cannot be performed (peripheral lesions) or creates a danger to the patient (iatrogenic hemorrhage).