Guidelines for the Cytopathologic Diagnosis of Epithelioid and Mixed-Type Malignant Mesothelioma: a secondary publication.

OBJECTIVE: To provide practical guidelines for the cytopathologic diagnosis of malignant mesothelioma. DATA SOURCES: Cytopathologists with an interest in the field involved in the International Mesothelioma Interest Group (IMIG) and the International Academy of Cytology (IAC) contributed to this update. Reference material includes peer-reviewed publications and textbooks. RATIONALE: This article is the result of discussions during and after the IMIG 2012 conference in Boston, followed by thorough discussions during the 2013 IAC meeting in Paris. Additional contributions have been obtained from cytopathologists and scientists who could not attend these meetings, with final discussions and input during the IMIG 2014 conference in Cape Town.

Impact of repeated nicotine and alcohol coexposure on in vitro and in vivo chlorpyrifos dosimetry and cholinesterase inhibition.

Chlorpyrifos (CPF) is an organophosphorus insecticide, and neurotoxicity results from inhibition of acetylcholinesterase (AChE) by its metabolite, chlorpyrifos-oxon. Routine consumption of alcohol and tobacco modifies metabolic and physiological processes impacting the metabolism and pharmacokinetics of other xenobiotics, including pesticides. This study evaluated the influence of repeated ethanol and nicotine coexposure on in vivo CPF dosimetry and cholinesterase (ChE) response (ChE- includes AChE and/or butyrylcholinesterase (BuChE)). Hepatic microsomes were prepared from groups of naive, ethanol-only (1 g/kg/d, 7 d, po), and ethanol + nicotine (1 mg/kg/d 7 d, sc)-treated rats, and the in vitro metabolism of CPF was evaluated. For in vivo studies, rats were treated with saline or ethanol (1 g/kg/d, po) + nicotine (1 mg/kg/d, sc) in addition to CPF (1 or 5 mg/kg/d, po) for 7 d. The major CPF metabolite, 3,5,6-trichloro-2-pyridinol (TCPy), in blood and urine and the plasma ChE and brain acetylcholinesterase (AChE) activities were measured in rats. There were differences in pharmacokinetics, with higher TCPy peak concentrations and increased blood TCPy AUC in ethanol + nicotine groups compared to CPF only (approximately 1.8- and 3.8-fold at 1 and 5 mg CPF doses, respectively). Brain AChE activities after ethanol + nicotine treatments showed significantly less inhibition following repeated 5 mg CPF/kg dosing compared to CPF only (96 ± 13 and 66 ± 7% of naive at 4 h post last CPF dosing, respectively). Although brain AChE activity was minimal inhibited for the 1-mg CPF/kg/d groups, the ethanol + nicotine pretreatment resulted in a similar trend (i.e., slightly less inhibition). No marked differences were observed in plasma ChE activities due to the alcohol + nicotine treatments. In vitro, CPF metabolism was not markedly affected by repeated ethanol or both ethanol + nicotine exposures. Compared with a previous study of nicotine and CPF exposure, there were no apparent additional exacerbating effects due to ethanol coexposure.

Pathological confirmation of primary lung cancer following breast cancer.

PURPOSE: Studies have shown that women who survive breast cancer have an increased risk of a future primary lung cancer, though many are based only on data recorded in tumor registries and none have conducted pathological confirmation. Previous studies and future use of large registries may be affected by misdiagnosis. METHODS: Pathological analysis was conducted on tumors from 110 women with breast cancer followed by lung cancer using morphology, Estrogen Receptor-alpha (ER), and Thyroid Transcription Factor-1 (TTF1). We developed an algorithm to classify lung tumors as unlikely lung cancer (score=1) to likely lung cancer (score=5). RESULTS: Mean time to diagnosis of lung cancer after breast cancer was 13 years. 76% of breast tumors and 20% of lung tumors were positive for ER and 51% of lung tumors were positive for TTF-1. 86% of the lung tumors were probable primaries, 7% were probable metastases from the breast, and 7% were of undetermined status. 70% of probable metastases had a latency of longer than 10 years. CONCLUSION: Prior studies identifying the association of breast cancer and breast cancer treatments with lung cancer are likely to reflect true associations not confounded by misdiagnosis, as evidenced by the low rate of misclassification detected in this study. Analysis of the years of diagnosis suggests that latency may not be an accurate criterion for assignment of primary status, which could be significant in a clinical setting. These data may also benefit future retrospective studies using large registries.

Age-specific prevalence of HPV genotypes in cervical cytology samples with equivocal or low-grade lesions.

BACKGROUND: To define the spectrum of human papillomavirus (HPV) types and establish an age limit for triage HPV testing in atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL). MATERIALS AND METHODS: 343 liquid-based cytological samples from the population-based screening programme with minor abnormalities were subjected to HPV genotyping (Linear Array, Roche, Basel, Switzerland). RESULTS: High-risk human papillomavirus (HR-HPV) was found in 71% of LSIL and 49% of ASCUS cases (P<0.001). High-risk human papillomavirus prevalence was age-dependent in LSIL (P=0.01), with decreasing prevalence until the age of 50 years, followed by a slight increase. Human papillomavirus type 16 was the most common HR-HPV, found in 23% of HPV-positive women. Human papillomavirus type 18 was the sixth most common, found in 9.9% (P<0.001). An age-dependent quadratic trend was observed for multiple infections (P=0.01) with a trough at about 42 years. The most common HR-HPV types to show a coinfection with HPV16 (clade 9) were HPV39 (28%), 45 (38%), and 59 (46%), belonging to HPV18 clade 7. The frequency of low-risk (LR) vs probable HR and HR-HPV also followed an age-dependent quadratic trend. CONCLUSIONS: After the age of 25 years, HR-HPV prevalence is similar in LSIL and ASCUS cases, motivating a low age limit for triage HPV testing. Multiple infections and LR/HR-HPV dominance are age-dependent. Genotyping in longitudinal design is needed to elucidate the importance of multiple infections in cancer progression and in cross-protection from vaccination.

Human papillomavirus 'reflex' testing as a screening method in cases of minor cytological abnormalities.

The aim was to evaluate human papillomavirus (HPV) 'reflex genotyping' in cases of minor cytological abnormalities detected in the gynaecological screening programme in Stockholm, Sweden. Liquid-based cytology samples showing minor cytological abnormalities were analysed using HPV genotyping (Linear Array, Roche diagnostics). Colposcopically directed cervical biopsies were obtained and the HPV test results were correlated with the histological results. In all, 63% (70/112) of the samples were high-risk (HR) HPV (HR-HPV) positive. A statistically significant correlation was found between high-grade cervical lesions and HR-HPV (P=0.019), among which HPV 16, 18, and 31 were the most important. The negative predictive value of HR-HPV detection for histologically confirmed high-grade lesions was 100%. An age limit for HPV reflex testing may be motivated in cases of low-grade squamous intraepithelial neoplasia (LSIL), because of high HR-HPV prevalence among younger women. By using HPV reflex genotyping, additional extensive workup can safely be avoided in about 50% of all cases of atypical squamous cells of undetermined significance (ASCUS) and LSIL among women 30 years. This screening strategy could potentially reduce the total abnormal cytology-reporting rate in the Swedish screening programme by about 1% and provide more accurately directed follow-up, guided by cytological appearance and HPV test results.

Versican but not decorin accumulation is related to metastatic potential and neovascularization in testicular germ cell tumours.

AIMS: To investigate the expression of versican and decorin in patients with testicular germ cell tumours (GCTs) and to correlate this with the clinicopathological findings. Matrix proteoglycans versican and decorin are frequently overexpressed in various malignancies and are involved in the progression of cancer. METHODS AND RESULTS: Overexpression of versican and decorin was detected in GCTs by immunoblotting. Immunohistochemical staining for proteoglycans was performed on 71 cases of paraffin-embedded tissues. In most of the cases increased decorin and versican stromal staining was demonstrated. In both seminomas and non-seminomatous germ cell tumours (NSGCTs) strong staining of decorin was not found to be related to any of the clinicopathological variables. Accumulation of versican was found to be associated with vascular and lymphatic invasion, nodal metastasis and disease stage in seminomas and NSGCTs and, in addition, with tumour size and distant metastasis only in NSGCTs. Additionally, only the deposition of versican was linearly correlated with the number of microvessels in the tumour stroma in GCTs. CONCLUSIONS: Ectopic versican and decorin expression is a frequent feature in GCTs. Versican but not decorin accumulation in GCTs is related to metastatic potential and neovascularization and might be a useful marker for testicular malignancy.

Frequent gain of the human telomerase gene TERC at 3q26 in cervical adenocarcinomas.

The level of genomic amplification of the human telomerase gene TERC, which maps to chromosome band 3q26, was determined in primary cervical adenocarcinomas. Interphase nuclei prepared from archival material of 12 primary cervical adenocarcinomas, eight of which were human papillomavirus positive, were hybridised with a triple colour probe set specific for centromeres of chromosomes 3 and 7 and the TERC gene. We observed high proportions of nuclei with increased absolute copy numbers for TERC in all tumours (mean 3.3; range 2.3-5.2). Amplification of the human telomerase gene TERC is a consistent aberration in cervical adenocarcinomas. Therefore, application of our probe set may provide an objective genetic test for the assessment of glandular cells in Pap smears and hence for the diagnosis of cervical adenocarcinomas.

Immunization with specific polysaccharide antigen reduces alterations in corneal proteoglycans during experimental slime-producing Staphylococcus epidermidis keratitis.

PURPOSE: Staphylococcus epidermidis is a leading cause of bacterial keratitis associated with corneal damage. Corneal integrity is closely associated with matrix macromolecules, such as proteoglycans (PGs) and collagen. The aim of this study was to examine whether active immunization (AI) using a major immunogenic polysaccharide determinant of slime (20-kDa PS) as antigen, and passive immunization (PI) after administration of specific antibodies toward 20-kDa PS affect the distribution of PGs as well as corneal lesions in an experimental model of slime-producing S. epidermidis keratitis. METHODS: For AI, seven rabbits were immunized with 20-kDa PS, whereas for PI, seven rabbits received specific antibodies against 20-kDa PS. Lesions were graded clinically for a 21-day period. Levels of 20-kDa PS antibodies in serum and aqueous humor in both immunization groups were determined by ELISA. The distribution of certain extracellular matrix PGs during corneal healing was analyzed immunohistochemically. RESULTS: Levels of specific anti-20-kDa PS antibodies in serum and aqueous humor obtained after either AI or PI were significantly higher as compared with those in the respective nonimmunized control groups (p<0.001). Clinical grading showed that both AI and PI rabbits had a significantly less corneal damage as compared with infected nontreated rabbits. Immunohistochemical analyses for PGs exhibited significant differences to the wounded regions as compared with noninfected corneal tissue. Accumulation of keratan sulfate PGs and decorin was observed in the corneal stroma of infected rabbits and of heparan sulfate PGs around the new-formed vessels. This phenomenon was significantly reduced in immunized animals in accordance with macroscopically decreased corneal damage observed in these animals. CONCLUSIONS: Results of this study suggest a key role of 20-kDa PS and its antibodies as prophylactic and therapeutic agents in keratitis caused by slime-producing S. epidermidis.

Expression of proteoglycan mRNA in patients with painful clicking and chronic closed lock of the temporomandibular joint.

RT-PCR was used to analyze the expression of a series of mRNAs coding for proteoglycans aggrecan, versican, biglycan, decorin, fibromodulin and also hyaluronan synthase 1 in specimens obtained during discectomy of the temporomandibular joint in patients with unilateral signs and symptoms of chronic closed lock (eight patients) and painful clicking (seven patients). Regarding the disc, aggrecan expression was higher in patients with chronic closed lock. As for the posterior disc attachment specimens, patients with chronic closed lock showed a tendency for higher expression of biglycan and hyaluronan synthase 1. The degradation of matrix in patients with chronic closed lock of the temporomandibular joint seems not to be caused by a reduced synthesis and the degenerative process seen in these patients is one with low turnover similar to the situation in primary osteoarthrosis of hyaline cartilage. The results indicate that any treatment should intervene early in the disease process of chronic closed lock in order to prevent the development of a degenerative process.

Versican undergoes specific alterations in the fine molecular structure and organization in human aneurysmal abdominal aortas.

Versican is the major matrix proteoglycan in aortic wall and participates in various biological functions of the tissue. In the present study the molecular characteristics of versican isolated from normal human aorta as well as those of versican expressed in aneurysmal aortic tissue were examined. Versican was isolated by combined anion-exchange and gel permeation chromatography and was further characterized by high-performance liquid chromatography, polyacrylamide gel electrophoresis and immunoblotting. In both tissues versican is exclusively substituted with chondroitin sulfate chains, in contrast to other human tissues where both chondroitin and dermatan sulfate chains are attached onto versican core proteins. Except for the significant decrease in the concentration of versican in the aneurysmal tissue, this PG undergoes specific alterations in the aneurysmal tissue. The molecular size of versican isolated from diseased tissue is decreased with a simultaneous increase in the ratio of glycosaminoglycan to protein in this tissue. The latter reflect the extensive fragmentation of versican in the diseased tissue and most probably the generation of shorter peptides enriched to glycosaminoglycan chains. Although the size of chondroitin sulfate chains is identical in both versican preparations, a significant increase in the percentage of 6-sulfated disaccharides is observed in chondroitin sulfate chains of versican in aneurysmal aortas, which is accompanied by decrease in 4-sulfated and non-sulfated units.

Matrix glycosaminoglycans in the temporomandibular joint in patients with painful clicking and chronic closed lock.

The aim was to investigate the content of 4- and 6-sulphated glycosaminoglycans (GAGs) in specimens from temporomandibular joint disc and posterior disc attachment in patients with painful clicking and chronic closed lock. Nineteen patients (19 joints) with a clinical diagnosis of painful clicking were compared with 22 patients (22 joints) with a clinical diagnosis of chronic closed lock. Specimens were obtained from the disc and the posterior disc attachment, and their content of glycosaminoglycans analysed by means of capillary zone electrophoresis. These were significant differences in the amount of glycosaminoglycans between the two groups, values in patients with painful clicking being comparable to those of normal individuals, while patients having chronic closed lock showed significantly reduced values. Both groups showed higher values in the posterior disc attachment when compared to the disc and similar pattern of glycosaminoglycan sulphation. The results suggest that these two patient groups have distinctly different patterns of tissue reactions. In patients with chronic closed lock there was an altered composition of matrix, this change involving both disc and posterior disc attachment.

Diagnosis of biliary strictures in conjunction with endoscopic retrograde cholangiopancreaticography, with special reference to patients with primary sclerosing cholangitis.

BACKGROUND AND STUDY AIMS: Strictures of the bile ducts due to malignant changes are difficult to distinguish from benign changes, particularly in patients with primary sclerosing cholangitis (PSC). The aim of this study was to evaluate diagnostic methods for malignancy in biliary strictures in conjunction with endoscopic retrograde cholangiopancreaticography (ERCP). PATIENTS AND METHODS: Bile duct strictures were identified during ERCP in 57 patients, who were thus included in the present study. Brush samples from the strictures were taken for cytology and for evaluation of DNA content by flow cytometry. The tumor markers CA 19-9 and CEA were determined both in serum and bile fluid. Two independent radiologists evaluated all cholangiograms. The diagnostic sensitivity, specificity, and accuracy of each diagnostic method were evaluated separately and in combination. RESULTS: 32 patients were found to have malignant strictures and when the four methods: brush cytology, DNA analysis, serum CA 19-9 and serum CEA were combined, a diagnostic sensitivity of 88 % and specificity of 80 % were reached. Seven of the 20 patients with PSC were found also to suffer from cholangiocarcinoma, yielding a sensitivity and specificity of 100 % and 85 %, respectively. Analyses of CA 19-9 and CEA in bile fluid had no diagnostic significance. CONCLUSION: An ERCP procedure with brush cytology, a DNA analysis, combined with serum analysis of CA 19-9 and CEA, can increase the possibility of distinguishing between malignant and benign biliary strictures, especially in PSC patients.

Analysis of glycosaminoglycan-derived disaccharides in biologic samples by capillary electrophoresis and protocol for sequencing glycosaminoglycans.

Glycosaminoglycans are biologically significant carbohydrates which either as free chains (hyaluronan) or constituents of proteoglycans (chondroitin/dermatan sulfates, heparin, heparan sulfate and keratan sulfate) participate and regulate several cellular events and (patho)physiological processes. Capillary electrophoresis, due to its high resolving power and sensitivity, has been successfully used for the analysis of glycosaminoglycans. Determination of compositional characteristics, such as disaccharide sulfation pattern, is a useful prerequisite for elucidating the interactions of glycosaminoglycans with matrix effective molecules and, therefore, essential in understanding the biological functions of proteoglycans. The interest in the field of characterization of such biologically important carbohydrates is soaring and advances in this field will signal a new revolution in the area of glycomics equivalent to that of genomics and proteomics. This review focuses on the capillary electrophoresis methods used to determine the disaccharide pattern of glycosaminoglycans in various biologic samples as well as advances in the sequence analysis of glycosaminoglycans using both chromatographic and electrophoretic techniques.

Value of carcinoembryonic antigen (CEA) and cholesterol assays of ascitic fluid in cases of inconclusive cytology.

AIM: To determine whether assays of carcinoembryonic antigen (CEA) and cholesterol in ascites add diagnostic value to cytology. METHODS: The additional diagnostic efficacy of the biochemical assays was studied in the ascitic fluid from 130 patients, of whom 57 had peritoneal carcinomatosis. All diagnoses were verified by subsequent necropsy and/or histology. RESULTS: CEA concentrations over 5 ng/ml indicated carcinomas, occasionally without peritoneal involvement of the tumour. However, increased values were significantly more common in cancer with peritoneal involvement (p < 0.01), giving a sensitivity of 51% and specificity of 97% for carcinomatosis. A cholesterol value exceeding 1.21 mmol/litre was found in 93% of cancers with peritoneal involvement, but it was not entirely specific (96%) for carcinomatosis. Simultaneous increases in CEA and cholesterol concentrations were specific for carcinomatosis and this combination increased the sensitivity for diagnosing carcinomatosis from 77% with cytology alone to 88%. The correct diagnosis could thus be made in five of 12 cases with inconclusive cytology. CONCLUSIONS: The measurements of both CEA and cholesterol concentrations in ascites give additional specific information about peritoneal carcinomatosis and can therefore be a useful adjunct to cytology-in particular, in inconclusive cases.

Microemulsion electrokinetic capillary chromatography of sulfated disaccharides derived from glycosaminoglycans.

Microemulsion electrokinetic capillary chromatography (MEEKC) is a capillary electrophoresis technique in which neutral and ionized species can be resolved according to their partitioning into moving oil droplets present in the operating buffer. In this report, we present for the first time the application of MEEKC in the analysis of glycosaminoglycans. An efficient method for the separation of the variously sulfated delta-disaccharides obtained following digestion of chondroitin and dermatan sulfates with chondro/ dermato lyases and derivatization with 2-aminoacridone is described. Nonsulfated, mono-, di-, and trisulfated delta-disaccharides were completely separated using the microemulsion octane/butan-1-ol/Sodium dodecyl sulfate (SDS) in 10 mM borate buffer, pH 9.3, at 25 kV. Agreement of the obtained disaccharide composition with literature values showed that MEEKC can be used for the analysis of glycosaminoglycans.

Synthesis and distribution of glycosaminoglycans in human leukemic B- and T-cells and monocytes studied using specific enzymic treatments and high-performance liquid chromatography.

Identification of glycosaminoglycans (GAGs) synthesized by three human leukaemic cell lines-Jurkat (T-cell leukaemia), Daudi (Burkitt's lymphoma, B-cell leukaemia) and THP-1 (acute monocytic leukemia)-and normal peripheral blood mononuclear cells (PBMC) and their distribution among cell membrane and culture medium were studied. GAGs were isolated using ion-exchange chromatography on DEAE-Sephacel and their composition and fine chemical structure were studied using high-performance liquid chromatography with radiochemical detection. All cell lines synthesize chondroitin sulphate (CS) and heparan sulphate (HS) in both cell membrane and culture medium. No hyaluronan was detected using treatment with specific lyases and highly sensitive HPLC methodology. CS is the major secreted GAG in all cell lines tested and the major cell retained GAG in Jurkat and Daudi. HS is the major GAG in the cell membrane of THP-1. The amounts of distinct GAGs synthesized by all cancer cell lines differ from those produced by normal PBML indicating a major role of GAGs in malignant transformation of human lymphocytes and monocytes.

Hyaluronan content in pleural fluid as a prognostic factor in patients with malignant pleural mesothelioma.

BACKGROUND: Regardless of the modality of therapy used, malignant pleural mesothelioma is a highly treatment-resistant and invariably fatal disease. Identification of prognostic variables are important for future investigational therapeutic studies. METHODS: The prognostic significance of various clinical variables, including hyaluronan levels in pleural fluid, was evaluated in a retrospective analysis in 100 patients with histologically confirmed malignant pleural mesothelioma. RESULTS: The overall median survival was 11.5 months. Univariate analyses identified histologic subtype, i.e., epithelial or mixed, and elevated content of hyaluronan in the pleural effusion as significant prognostic variables. A multivariate analysis confirmed the independent predictive power of histologic subtype, and an elevated concentration of hyaluronan in the pleural fluid also indicated longer survival in older patients and in patients receiving therapy other than supportive. CONCLUSIONS: The prognostic value of histologic subtype and the concentration of hyaluronan in pleural effusions should be considered when designing and evaluating treatment trials for patients with malignant pleural mesothelioma.

Identification of oligomeric domains within dermatan sulfate chains using differential enzymic treatments, derivatization with 2-aminoacridone and capillary electrophoresis.

Galactosaminoglycans, i.e. dermatan sulfate (DS) and chondroitin sulfate, are linear heteropolysaccharides consisting of repeating disaccharide units of L-iduronic acid (L-IdoA) or D-glucuronic acid (D-GlcA) residues linked to N-acetyl-galactosamine. High-performance capillary electrophoresis (HPCE or CE) has been successfully used for determining the disaccharide composition of glycosaminoglycans. However, only limited information is available on how to identify oligomeric domains rich in D-GlcA or L-IdoA. The aim of this study was therefore to develop a rapid and accurate CE procedure by which such oligosaccharides can be determined together with the variously sulfated disaccharides. Isolated dermatan sulfates of human origin were separately digested with chondroitinases ABC, AC and B and the enzymic products were derivatized with 2-aminoacridone. CE analysis of these products was performed using a phosphate buffer, pH 3.0, and reversed polarity at 30 kV. The derivatization enabled their detection with laser-induced fluorescence (LIF) and UV at 260 nm at much higher sensitivity than the detection of nonderivatized delta-saccharides at 232 nm and therefore components undetectable at 232 nm were nicely detected after derivatization. Except for delta-disaccharides, altogether five distinct oligosaccharides with differences in charge density were identified. Depending on the lyase that produced these oligomers, information on the presence of L-IdoA- or D-GlcA-containing domains within the DS chain and the sulfation pattern of these oligomeric domains was obtained. This CE method could also be useful in studying the functional oligomeric domains in galactosaminoglycan chains.