Combined assessment of polymorphisms in the LHCGR and FSHR genes predict chance of pregnancy after in vitro fertilization.

STUDY QUESTION: Can gonadotrophin receptor variants separately or in combination, be used for the prediction of pregnancy chances in in vitro fertilization (IVF) trials? SUMMARY ANSWER: The luteinizing hormone/human chorionic gonadotrophin receptor (LHCGR) variant N312S and the follicle-stimulating hormone receptor (FSHR) variant N680S can be utilized for the prediction of pregnancy chances in women undergoing IVF. WHAT IS KNOWN ALREADY: The FSHR N680S polymorphism has been shown to affect the ovarian response in response to gonadotrophin treatment, while no information is currently available regarding variants of the LHCGR in this context. STUDY DESIGN, SIZE, DURATION: Cross-sectional study, duration from September 2010 to February 2015. Women undergoing IVF were consecutively enrolled and genetic variants compared between those who became pregnant and those who did not. The study was subsequently replicated in an independent sample. Granulosa cells from a subset of women were investigated regarding functionality of the genetic variants. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women undergoing IVF (n = 384) were enrolled in the study and genotyped. Clinical variables were retrieved from medical records. For replication, an additional group of n = 233 women was utilized. Granulosa cells from n = 135 women were isolated by flow cytometry, stimulated with Follitropin alpha or Menotropin, and the downstream targets 3',5'-cyclic adenosine monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP3) measured with enzyme-linked immunosorbent assay. MAIN RESULTS AND THE ROLE OF CHANCE: Women homozygous for serine (S) in both polymorphisms displayed higher pregnancy rates than women homozygous asparagine (N) (OR = 14.4, 95% CI: [1.65, 126], P = 0.016). Higher pregnancy rates were also evident for women carrying LHCGR S312, regardless of FSHR variant (OR = 1.61, 95% CI: [1.13, 2.29], P = 0.008). These women required higher doses of FSH for follicle recruitment than women homozygous N (161 versus 148 IU, P = 0.030). When combining the study cohort with the replication cohort (n = 606), even stronger associations with pregnancy rates were noted for the combined genotypes (OR = 11.5, 95% CI: [1.86, 71.0], P = 0.009) and for women carrying LHCGR S312 (OR = 1.49, 95% CI: [1.14, 1.96], P = 0.004). A linear significant trend with pregnancy rate and increasing number of G alleles was also evident in the merged study population (OR = 1.34, 95% CI: [1.10, 1.64], P = 0.004). A lower cAMP response in granulosa cells was noted following Follitropin alpha stimulation for women homozygous N in both polymorphisms, compared with women with other genotypes (0.901 pmol cAMP/mg total protein versus 2.19 pmol cAMP/mg total protein, P = 0.035). LIMITATIONS, REASONS FOR CAUTION: Due to racial differences in LHCGR genotype distribution, these results may not be applicable for all populations. WIDER IMPLICATIONS OF THE FINDINGS: Despite that >250 000 cycles of gonadotrophin stimulations are performed annually worldwide prior to IVF, it has not been possible to predict neither the pregnancy outcome, nor the response to the hormone with accuracy. If LHCGR and FSHR variants are recognized as biomarkers for chance of pregnancy, more individualized and thereby more efficient treatment modalities can be developed. STUDY FUNDING, COMPETING INTERESTS: This work was supported by Interreg IV A, EU (grant 167158) and ALF governments grant (F2014/354). Merck-Serono (Darmstadt, Germany) supported the enrollment of the subjects. The authors declare no conflict of interest.

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.

In situ telomerase activity in pleural effusions: a promising marker for malignancy.

Telomerase activity in 16 pleural effusions was studied using an in situ telomerase repeat amplification protocol (TRAP) assay on cytospin preparations. Six of nine cytologically malignant specimens contained telomerase-positive cells (67%), and in two further specimens, suspicious positive cells were seen. Two of four atypical specimens contained telomerase-positive cells, whereas two benign cases were telomerase-negative. No mesothelial cells showed telomerase reactivity. Thus, telomerase activity was specific for malignancy and it was always found only in malignant cells. The results suggest that telomerase activity measured with this in situ method can be a valuable complement in the assessment of malignancy in pleural effusions.

An optimized battery of eight antibodies that can distinguish most cases of epithelial mesothelioma from adenocarcinoma.

An immunocytochemical battery comprising 9 antibodies specifically distinguishes 80% of the epithelial malignant mesotheliomas from adenocarcinomas. The discriminatory power of antibodies to calretinin was tested together with this battery to determine whether the performance thereby could be improved. The study comprises 119 mesotheliomas of epithelial or mixed phenotype and 57 adenocarcinoma metastases in the pleural cavity. The differences between the 2 groups were highly significant for all recorded parameters, but typical reactivity for all parameters was seen in only 6 (5.0%) of the 119 mesotheliomas. An algorithm based on stepwise logistic regression was used to interpret divergent reaction patterns. Most diagnostic information was obtained with 8 of the parameters studied. The resulting algorithm identified almost 90% of the mesotheliomas with high specificity. The battery can be performed in 2 steps: several adenocarcinomas first are diagnosed with a few antibodies, applying the rest of the battery on the remaining unresolved cases.

Correlation between morphology and telomerase activity in cells from exfoliative lung cytologic specimens.

BACKGROUND: Telomerase is a ribonucleoprotein that compensates for the erosion of telomeres (chromosomal termini). Telomerase activity is detected in more than 85% of cancerous lesions and is therefore considered a novel marker of cancer. The authors compared cytologic morphology and telomerase activity at the cellular level to obtain further insight into their association. METHODS: The authors used bronchial washing and brushing materials obtained from 18 patients with lung carcinomas (6 squamous cell, 8 adenocarcinoma, 2 large cell, 1 small cell, and 1 metastasis from colon carcinoma) and 20 patients with nonmalignant disease. An in situ telomeric repeat amplification protocol (TRAP) assay was performed, and routine Papanicolaou-stained slides using the same sample were assessed. RESULTS: Nuclear fluorescent signals at the nuclear area, corresponding to telomerase activity, shown by the in situ TRAP assay were only detected in samples containing morphologically malignant cells. No nuclear fluorescence was seen in the keratinizing component of well-differentiated squamous cell carcinoma. Nuclear staining was not seen in metaplastic or basal hyperplastic cells. Cytoplasmic fluorescence was only found in macrophages and polymorphonuclear leukocytes. CONCLUSIONS: Nuclear fluorescence corresponding to telomerase activity was not demonstrated in metaplastic or basal hyperplastic cells, thus indicating that detection of telomerase activity is closely associated with the presence of malignant cells, but not premalignant lesions, in lung carcinoma patients. Moreover, in some samples with cancer, cells failed to show telomerase activity, suggesting the limitation of this method for the detection of malignant cells in certain lung carcinoma patients.

Reactivity of six antibodies in effusions of mesothelioma, adenocarcinoma and mesotheliosis: stepwise logistic regression analysis.

Anti-CEA, anti-vimentin, CAM5.2, BerEp4, Leu-M1 and anti-EMA were applied to effusions from 36 mesotheliomas, 53 adenocarcinomas and 24 reactive mesothelial proliferations. Stepwise logistic regression analysis selected three criteria of major importance for distinguishing between adenocarcinoma and mesothelioma: BerEp4, CEA and EMA accentuated at the cell membrane (mEMA), these three being of similar diagnostic value. The pattern BerEp4-, CEA- and mEMA+ was fully predictive for mesothelioma (sensitivity 47%), whereas the opposite pattern was fully predictive for adenocarcinoma (sensitivity 80%). Only EMA seemed to distinguish between mesotheliosis and mesothelioma. Comparison of reactivity in cytological and histological material from the same mesotheliomas showed similar staining frequencies for CEA and CAM5.2, with some random variation for Leu-M1 and EMA, whereas vimentin and BerEp4 reactivity was more frequent in cytological specimens.

Comparison of Papnet-assisted and manual screening of cervical smears.

We compared the performance of Papnet to manual screening with respect to detection of dysplastic/atypical epithelial cells, endocervical cells, and inflammatory changes/infectious agents. One thousand consecutive manually screened smears were retrieved from the cytological files and subjected to Papnet analysis. Five slides had to be excluded for technical reasons. Of the remaining 995 cases, manual and Papnet diagnosis showed agreement in 986 cases, 950 benign or within normal limits, and 36 with epithelial abnormalities. Papnet detected 4 cases missed by manual screening, but failed to detect 5 manually diagnosed cases. In 293/749 smears, existing endocervical cells were not selected by Papnet (40%) and inflammatory changes/infectious agents were diagnosed in 49 cases, compared to 87 cases by manual screening (56%). Papnet equaled manual screening for dysplasias and epithelial atypias, but its poor performance regarding endocervical cells and inflammation would be a drawback in primary screening. Diagn. Cytopathol. 1999;21:296-299.

Immunoreactivity of pleural malignant mesotheliomas to glutathione S-transferases.

Malignant mesotheliomas show a highly variable aggressiveness, but it is difficult to predict the outcome in the individual case at the time of diagnosis. Glutathione S-transferases are detoxification enzymes that have been correlated with the prognosis in some tumours. We have therefore assessed the value of GST expression as a prognostic parameter in mesotheliomas. The reactivities to GST-pi, -alpha and -mu antibodies were studied in histological sections from altogether 88 cases. Most of them showed distinct cytoplasmic reactivity to one or more of the GST antibodies tested. This high prevalence is in good agreement with the low responsiveness of mesotheliomas to chemotherapy. However, there was no prognostic value in detecting GST immunoreactivity, and it gave no information of value for distinguishing between neoplastic and reactive mesothelium.

Optimization of a battery using nine immunocytochemical variables for distinguishing between epithelial mesothelioma and adenocarcinoma.

A battery of immunocytochemical analyses, previously established to distinguish between malignant mesothelioma and metastatic adenocarcinoma, was extended by analysing the same cases with three other commercially available antibodies. Altogether, 11 antibodies were studied in mesotheliomas diagnosed by other means, using 14 different immunocytochemical parameters. Logistic regression analysis indicated that the following parameters were of importance for this diagnostic problem: vimentin reactivity in epithelial cells (1), cytokeratin (CAM 5.2) reactivity in spindle-shaped (fibrous) cells (2), cell membrane-associated reactivity of EMA (3), HBME-1 (4) and thrombomodulin (5), and absence of reactivity to CEA (6), CD15 (7), BerEp4 (8) and Sialyl-TN (9). The analysis gave an algorithm with which a specific diagnosis of mesothelioma could be made in 80% of the cases-i.e., some improvement compared to the 55% sensitivity using the previous battery.

Bilateral isolated adrenal coccidioidomycosis.

A case of bilateral isolated adrenal coccidioidomycosis in a previously healthy and immunocompetent 43-year-old Caucasian male is presented. He had never travelled to a coccidioidomycosis endemic area. Subclinical adrenal dysfunction was found with elevated plasma ACTH and mineralocorticosteroid and androgen pathway abnormalities. The implement of the fungal infection on adrenal function, and the diagnosis and management of adrenal coccidioidomycosis are discussed.

Clinical utility of liquid-chromatographic analysis of effusions for hyaluronate content.

A previously described HPLC method for determining hyaluronate in effusions was used to analyze a consecutive series of effusions from 1039 patients with pleural fluids and from 571 patients with peritoneal fluids. A mesothelioma was verified histologically in 50 of the cases. The results were used to estimate the clinical utility of the analysis. With a cutoff of 75 mg/L for hyaluronate-derived uronic acid, assay specificity for a malignant mesothelioma was 100% and the sensitivity 56%. Only 20% of the effusions from the mesothelioma patients showed no evidence of increased production of hyaluronate. Cytological smears from the associated cell pellets were evaluated as malignant or suspicious for malignancy in only 28% or in a further 46% of the mesothelioma cases, respectively, leaving 30% of the pellets as cytologically false-negative. We also analyzed effusions from selected cases submitted from other hospitals, 154 of which had been diagnosed histologically as mesotheliomas. Concentrations of hyaluronate were increased in these cases too, but a considerable proportion of the samples showed evidence of losses of hyaluronate; consequently, the sensitivity of the assay in these samples was lower.

Immunohistochemical reactivity in mesothelioma and adenocarcinoma: a stepwise logistic regression analysis.

Histological sections from 103 malignant mesotheliomas and 43 adenocarcinoma metastases in pleural biopsies were investigated for reactivity against a panel of 11 different antibodies. The size of the material allowed the evaluation by stepwise logistic regression analysis, which selected five parameters of major importance: vimentin reactivity in epithelial cells, reactivity to low-molecular-weight keratins in fibrous cells, strong membrane accentuation of EMA reactivity, and lack of reactivity to LeuM1 and BerEp4. Three of these criteria were sufficient to identify a mesothelioma with high specificity and with a sensitivity of approximately 70%. Whilst the monoclonal anti-CEA tested was the most valuable single parameter, it did not add any diagnostic information to the combination of criteria selected by the stepwise logistic regression analysis. However, this antibody can be used to exclude most of the adenocarcinomas from further analysis with the more extensive panel.

Carcinoembryonic antigen-like reactivity in malignant mesothelioma. A comparison between different commercially available antibodies.

BACKGROUND: Immunohistochemical reactivity to carcinoembryonic antigen (CEA) is often used to exclude the diagnosis of malignant mesothelioma. METHODS: In a study comprising 61 malignant mesotheliomas and 20 adenocarcinomas, the reactivity against one polyclonal and five different monoclonal anti-CEA antibodies was tested. RESULTS: Thirteen of the mesotheliomas showed distinct reactivity to the polyclonal antiserum, even after absorption with spleen powder. Of the monoclonal preparations, those staining granulocytes and macrophages also showed varying reactivity to some of these 13 mesotheliomas. Only one monoclonal antibody showed no such reactivity and labeled no mesothelioma case, while its capacity to stain adenocarcinomas remained. CONCLUSIONS: Mesotheliomas may contain substances that share epitopes with the CEA molecule. Only one of the tested monoclonal antibodies seemed sufficiently specific to be used diagnostically as a single immunohistochemical parameter to exclude malignant mesothelioma.

Prognostic importance of the DNA ploidy pattern in malignant mesothelioma of the pleura.

Malignant mesotheliomas often raise a difficult diagnostic problem: once the diagnosis is made, the possibilities of predicting the biologic activity and prognosis of the tumor are limited. DNA ploidy patterns have been used as a prognostic instrument for other tumors, and this pattern was therefore studied in 37 cases of verified mesothelioma. The measurements were made on Feulgen-stained smears from pleural effusions using a Leica Miamed computer microscope. When a highly aneuploid tumor was defined as a condition with greater than 5% of the cells within defined intervals outside the ranges of the stem-line and the peaks representing polyploidization thereof, a near-diploid/polyploid pattern was obtained in 41% of the cases, while the remaining 59% were classified as highly aneuploid. The prognosis was significantly better among the near-diploid/polyploid cases. This estimate of the outcome could not be improved by using alternative algorithms for high-grade aneuploidy.

Gaucher's disease in an infant diagnosed by fine needle aspiration of the liver and spleen. A case report.

A case of Gaucher's disease diagnosed by means of fine needle aspiration of the liver and spleen in a 12-month-old boy with hepatosplenomegaly is presented. The diagnosis was based on the finding of large, macrophagelike cells with abundant, pale, fibrillary cytoplasm and small nuclei. The patient had no family history of Gaucher's disease, and the diagnosis was not suspected clinically.

Frequency of cytologic features in fine needle aspirates from histologically and cytologically diagnosed fibroadenomas.

Identification of key cytologic criteria for various lesions by means of stepwise logistic regression is common today and is often claimed to facilitate cytologic diagnosis. As can be expected, there usually is good agreement between the selected criteria and those given in the cytologic literature. In a study by Bottles et al based on 64 fibroadenomas, stroma, antler horn clusters and honeycomb sheets differentiated fibroadenoma from ductal carcinoma. We applied Bottles's criteria to our fibroadenoma material in order to test their value in clinical cytologic practice. In fine needle aspirates from histologically (cytologically) diagnosed fibroadenomas, stroma was found in 41 (57%), antler horn clusters in 59 (90%) and honeycomb sheets in 62 (81%), which reduced the clinical value of the criteria.

Fine needle aspiration biopsy of cystic lesions of the head and neck, excluding the thyroid.

The occurrence of cystic lesions of the head and neck region (excluding the thyroid gland) and their diagnosis by fine needle aspiration (FNA) biopsy were reviewed for a two-year period. Of the 967 total aspirates of the region, 98 were cystic. The frequency of cysts was thus 10% in the total material, including 23% in parotid gland aspirates and 3% in lymph node aspirates. Salivary gland cysts occurred more often on the right side. The overall malignancy rate was the same for cysts and solid lesions (16%); however, 81% of cystic lymph node lesions were malignant. There was no difference in the frequency of nondiagnostic FNA material between solid and cystic lesions. A histologically correct diagnosis of benign or malignant was rendered by cytology in 85% of the cystic cases, with 4% false negatives and 2% false positives. This diagnostic accuracy was similar to that of the solid lesions. The FNA diagnostic problems were concentrated in the salivary gland lesions, with the false diagnoses illustrating the difficulties of interpreting atypical oxyphilic epithelium. The results suggest that a true neoplastic lesion should be seriously considered when a single population of oxyphilic epithelium is identified, even at the risk of overdiagnosing benign lesions.

Muco-epidermoid tumour of the bronchus.

A 7-year-old boy with recurrent pneumonia of the right lower lobe is described. At bronchoscopy a small tumour almost totally obliterating the right lower lobe was detected. Surgical treatment was undertaken. Histopathologic studies showed findings consistent with those of a muco-epidermoid tumour. The case emphasizes bronchoscopy as an important investigation in children with recurrent pneumonia. It also shows the importance of performing the bronchoscopy during a prolonged course of antibiotic prophylaxis in order to ensure a minimum of infected mucus within the bronchi. This report is completed with a short review of the literature on muco-epidermoid tumours of the bronchus in children. Their favourable prognosis and very low malignant potential is underlined.