Semiautomated quantitative image analysis of glomerular immunohistochemistry markers desmin, vimentin, podocin, synaptopodin and WT-1 in acute and chronic rat kidney disease models.

Five different glomerular immunohistochemistry markers were evaluated and compared in four different acute and chronic rat kidney disease models. Progression of glomerular or podocyte damage was shown in the puromycin aminonucleoside nephrosis (PAN) and Zucker fatty/spontaneously hypertensive heart failure F1 hybrid (ZSF1) rat model. Progression and prevention of glomerular damage was demonstrated in the Zucker diabetic fatty (ZDF) and Dahl salt-sensitive (Dahl SS) rat. Immunohistochemistry was performed for desmin, vimentin, podocin, synaptopodin and Wilms tumor protein-1 (WT-1), and evaluation of glomerular immunohistochemistry markers was done by semiautomated quantitative image analysis. We found desmin and WT-1 as the most sensitive markers for podocyte damage in both acute and chronic glomerular damage followed by vimentin, podocin and synaptopodin. We were able to demonstrate that early podocyte damage as shown by increased desmin and vimentin staining together with either a phenotypic podocyte change or podocyte loss (reduced numbers of WT-1-stained podocytes) drives the progression of glomerular damage. This is followed by a reduction in podocyte-specific proteins such as podocin and synaptopodin. Our report describes the different sensitivity of glomerular or podocyte markers and gives future guidance for the selection of the most sensitive markers for efficacy testing of new drugs as well as for the selection of tissue-based toxicity markers for glomerular or podocyte injury. In addition to functional clinical chemistry markers, desmin and WT-1 immunohistochemistry offers reliable and valuable data on the morphologic state of podocytes.

Somatic copy number changes in DPYD are associated with lower risk of recurrence in triple-negative breast cancers.

BACKGROUND: Genomic rearrangements at the fragile site FRA1E may disrupt the dihydropyrimidine dehydrogenase gene (DPYD) which is involved in 5-fluorouracil (5-FU) catabolism. In triple-negative breast cancer (TNBC), a subtype of breast cancer frequently deficient in DNA repair, we have investigated the susceptibility to acquire copy number variations (CNVs) in DPYD and evaluated their impact on standard adjuvant treatment. METHODS: DPYD CNVs were analysed in 106 TNBC tumour specimens using multiplex ligation-dependent probe amplification (MLPA) analysis. Dihydropyrimidine dehydrogenase (DPD) expression was determined by immunohistochemistry in 146 tumour tissues. RESULTS: In TNBC, we detected 43 (41%) tumour specimens with genomic deletions and/or duplications within DPYD which were associated with higher histological grade (P=0.006) and with rearrangements in the DNA repair gene BRCA1 (P=0.007). Immunohistochemical analysis revealed low, moderate and high DPD expression in 64%, 29% and 7% of all TNBCs, and in 40%, 53% and 7% of TNBCs with DPYD CNVs, respectively. Irrespective of DPD protein levels, the presence of CNVs was significantly related to longer time to progression in patients who had received 5-FU- and/or anthracycline-based polychemotherapy (hazard ratio=0.26 (95% CI: 0.07-0.91), log-rank P=0.023; adjusted for tumour stage: P=0.037). CONCLUSION: Genomic rearrangements in DPYD, rather than aberrant DPD protein levels, reflect a distinct tumour profile associated with prolonged time to progression upon first-line chemotherapy in TNBC.

Discovery of potent and selective histamine H3 receptor inverse agonists based on the 3,4-dihydro-2H-pyrazino[1,2-a]indol-1-one scaffold.

A novel series of potent histamine H(3) receptor inverse agonists based on the 3,4-dihydro-2H-pyrazino[1,2-a]indol-1-one scaffold has been discovered. Several compounds display high selectivity over other histamine receptor subtypes and have favorable physicochemical properties, low potential for CYP450 enzyme inhibition and high metabolic stability in microsomal preparations. (R)-2-Cyclopropylmethyl-8-(1-isopropyl-piperidin-4-yloxy)-3-methyl-3,4-dihydro-2H-pyrazino[1,2-a]indol-1-one (8t) showed good in vivo efficacy after per os application in an acute rat dipsogenia model of water intake.

A mutation in the AtPRP4 splicing factor gene suppresses seed development in Arabidopsis.

The spliceosome catalyzes alternative splicing of many genes in eucaryotic cells. This leads to the expression of distinct proteins. Components of the spliceosome are conserved in mammals and plants. Because splicing can be affected by environmental stress, we analyzed the regulation of splicing-related genes that encode small nuclear ribonucleoprotein particle (snRNP) proteins by the stress hormone abscisic acid (ABA). The transcript abundance of about 25 % of those genes was changed by at least 1.5-fold after addition of ABA. The U4/U6-specific snRNP gene AtPRP4 was strongly repressed by ABA. The homozygous knock-out of AtPRP4 resulted in the suppression of seed development suggesting that the gene product of this stress hormone-regulated gene is crucial for normal seed development.

Randomized trial of cyclophosphamide, doxorubicin, and 5-fluorouracil alone or alternating with a "cycle active" non-cross-resistant combination in women with visceral metastatic breast cancer: a Southeastern Cancer Study Group project.

Patients with visceral patterns of metastatic breast cancer were stratified according to dominant metastatic site and performance status and then randomized to therapy with cyclophosphamide, doxorubicin, and 5-fluorouracil (CAF) or CAF alternating with a "cell-cycle active" regimen including cytosine arabinoside, methotrexate with leucovorin rescue, and oncovin ( CAMELEON ). One hundred eighty-seven patients were randomized; response rate for CAF was 44% and for CAF + CAMELEON , 40%. Durations of disease control and survival were not significantly different. Toxicity of CAF was as anticipated with predominant granulocytopenia, vomiting, and alopecia. Toxicity of CAMELEON was less severe than that of CAF, and CAF toxicity was not worsened by preceding courses of CAMELEON ; however, the CAF- CAMELEON regimen was cumbersome and complex leading to both physician and patient noncompliance. Contrary to the preliminary results of a pilot study, and preliminary reports of the present trial suggesting benefit for the CAF- CAMELEON regimen the present randomized trial does not confirm any significant benefit of CAF- CAMELEON over CAF alone in patients with visceral metastatic breast cancer although this conclusion must be viewed in light of the high inevaluability rate due to patient and physician noncompliance.

Neutralization of (NK-cell-derived) B-cell activating factor by Belimumab restores sensitivity of chronic lymphoid leukemia cells to direct and Rituximab-induced NK lysis.

Natural killer (NK) cells are cytotoxic lymphocytes that substantially contribute to the therapeutic benefit of antitumor antibodies like Rituximab, a crucial component in the treatment of B-cell malignancies. In chronic lymphocytic leukemia (CLL), the ability of NK cells to lyse the malignant cells and to mediate antibody-dependent cellular cytotoxicity upon Fc receptor stimulation is compromised, but the underlying mechanisms are largely unclear. We report here that NK-cells activation-dependently produce the tumor necrosis factor family member 'B-cell activating factor' (BAFF) in soluble form with no detectable surface expression, also in response to Fc receptor triggering by therapeutic CD20-antibodies. BAFF in turn enhanced the metabolic activity of primary CLL cells and impaired direct and Rituximab-induced lysis of CLL cells without affecting NK reactivity per se. The neutralizing BAFF antibody Belimumab, which is approved for treatment of systemic lupus erythematosus, prevented the effects of BAFF on the metabolism of CLL cells and restored their susceptibility to direct and Rituximab-induced NK-cell killing in allogeneic and autologous experimental systems. Our findings unravel the involvement of BAFF in the resistance of CLL cells to NK-cell antitumor immunity and Rituximab treatment and point to a benefit of combinatory approaches employing BAFF-neutralizing drugs in B-cell malignancies.

Influence of major-histocompatibility-complex-compatible and incompatible Langerhans cells on the survival of H-Y-incompatible skin grafts in rats.

BN male trunk skin grafts, the Langerhans cells (LC) of which have been replaced with major histocompatibility complex (MHC)-incompatible Lewis female cells, survive longer on BN female rats than BN male trunk skin grafts bearing BN female LC. Evidence is presented that the privilege afforded these grafts is a consequence of MHC restriction. Thus, supplanting the native LC population of BN male trunk skin grafts with MHC-compatible Lewis.1N female cells has no affect on their survival on BN female rats. Evidence is also presented that H-Y-incompatible isografts deficient in MHC-compatible LC induce tolerance of H-Y.

Pediatric thyroid nodules: disease demographics and clinical management as determined by fine needle aspiration biopsy.

OBJECTIVE: The prevalence of thyroid nodularity in children has been estimated to be 1.8%. The reported prevalence of specific diseases which comprise these nodules is conflicting as evidenced by a reported range of malignancy of 2 to 50% in solitary nodules. In order to better classify pediatric (< 18 years old) thyroid disease and evaluate the utility of fine needle aspiration biopsy (FNAB) in this patient population, we retrospectively reviewed 66 FNABs from 64 thyroid nodules and 2 perithyroid lymph nodes from 57 patients. PATIENTS: The study was composed of 8 males and 49 females who ranged in age from 1 to 18 years old (mean = 13.1). DESIGN: Surgical and/or clinical follow-up was obtained in all patients. The 66 FNAB diagnoses were initially classified into specific diseases. However, for the purpose of this review, the cases were classified as: 3 insufficient, 51 benign, 8 suspicious, and 4 malignant. RESULTS: There were no "false positives" and one "false negative" (a papillary carcinoma was misdiagnosed as a benign nodule). Overall, 10 patients (18%) had malignant thyroid lesions, including 8 papillary carcinomas and 2 follicular carcinomas. Benign diagnoses included benign nodule, cyst, lymphocytic thyroiditis, granulomatous thyroiditis, hyperplasia, and abscess. CONCLUSIONS: The prevalence of malignancy in pediatric patients with thyroid nodules was 18%. We conclude that, because of its high diagnostic accuracy and minimal invasiveness, FNAB is useful in the management of pediatric thyroid nodules.

p53 protein overexpression in smooth muscle tumors of the uterus.

p53 is a nuclear phosphoprotein whose overexpression may portend a poor prognosis in a variety of neoplasms. In this immunohistochemical study we examined p53 overexpression in a variety of uterine smooth muscle tumors (34 leiomyosarcomas, 18 leiomyomas, and six smooth muscle tumors of uncertain malignant potential [STUMPs]). p53 immunoreactivity was observed in none of 18 (0%) leiomyomas, one of six (17%) STUMPs, and 16 of 34 (47%) leiomyosarcomas. Reactivity was not observed in the surrounding nonneoplastic uterine smooth muscle. Strong p53 overexpression in the leiomyosarcomas was significantly associated with high grade morphology (P = .013) and a high stage at the time of presentation (P = .021). In 25 leiomyosarcoma patients with clinical follow-up, p53 overexpression was associated with shorter length of survival (P = 0.024). However, this effect was not independent of tumor stage or grade. A regression analysis showed that tumor stage was the only independent predictor of length of survival. Our study size is small, and further studies are warranted to determine the significance and replicability of these findings.

The effect of a patient's risk-taking attitude on the cost effectiveness of testing strategies in the evaluation of pulmonary lesions.

STUDY OBJECTIVES: To assess the health and cost effects of a patient's risk-taking attitudes about diagnostic tests. DESIGN: Cost-effectiveness analysis. SETTING: Diagnostic testing strategies used in the evaluation of a patient with a radiographically detected lung lesion were evaluated. Strategies included combinations of sputum, fine-needle aspiration, bronchoscopy, thoracoscopy, and expectant management. PATIENTS: Patient data were obtained from the Survival Epidemiology and End Results Program, MEDLINE search, National Center for Health Statistics, and the Universities of Iowa and Stanford, and Kaiser Permanente Hospital. INTERVENTIONS: Different patient risk-taking attitudes were simulated using decision analysis. MEASUREMENTS: Lifetime cost of medical care, life expectancy, and cost effectiveness. RESULTS: The cost effectiveness of competing strategies depended on patient attitudes about taking risks. For a patient averse to expectantly waiting without definitive knowledge of whether cancer was or was not present, testing strategies using invasive procedures, such as thoracoscopy, were more cost effective. In contrast, for a patient who was identical except that he or she was averse to tests with higher morbidity and mortality, strategies that involved expectantly waiting, instead of more invasive tests, were more cost effective. Small changes in some risk-taking attitudes resulted in large changes in cost effectiveness. CONCLUSIONS: Risk-taking attitudes influenced the cost effectiveness of testing strategies. Consideration of patient risk-taking attitudes in diagnostic testing appears warranted in setting clinical policies and making individual decisions.

The importance of sputum cytology in the diagnosis of lung cancer: a cost-effectiveness analysis.

STUDY OBJECTIVE: To assess the potential health and cost effects of initial testing with sputum cytology to diagnose lung cancer. DESIGN: Cost-effectiveness analysis. DATA SOURCES: Surveillance Epidemiology and End Results (SEER) program; cost data from Northern California Kaiser Permanente Hospitals and Universities of Stanford and Iowa; National Center for Health Statistics; and a MEDLINE search. INTERVENTIONS: The use of sputum cytologies preceding other tests (ie, fine-needle aspiration, bronchoscopy, thoracoscopy) in patients with suspected lung cancer. MAIN OUTCOME MEASURES: Mortality associated with testing and initial surgical treatment (eg, performance of thoracoscopy to remove a local-stage, centrally located cancer), cost of testing and initial treatment, life expectancy, lifetime cost of medical care, and cost-effectiveness. RESULTS: In central lesions, sputum cytology as the first test was the dominant strategy because it both lowers medical-care costs ($2,516 per patient) and lowers the mortality risk (19 deaths in 100,000 patients) of the evaluation without adversely affecting long-term survival. In peripheral lesions, sputum cytology costs less then $25,000 per year of life saved if the pretest probability of cancer exceeds 50%. The estimated annual savings of adopting sputum cytology as the first test for diagnosing lung cancer in the United States is at least $30 million. CONCLUSIONS: Experience in regional centers indicates that sputum cytologic testing is infrequently ordered before implementing invasive diagnostic techniques, even in patients with central lung masses. The study findings suggest that sputum cytology as the first test in suspected lung cancer is likely to be cost saving without adversely affecting patient outcomes.

Subpopulations of lymphocytes in maternal peripheral blood during pregnancy.

The fetus can be considered an allograft with up to one-half of its MHC antigens being potentially recognized by the mother as foreign. This study compares expression of OKT3, OKT4, OKT8, Kappa, Lambda and Ia antigens on lymphocytes in the peripheral blood of normal non-pregnant women, normal pregnant women, patients who are chronic spontaneous aborters and pregnant insulin-dependent diabetic women. Monoclonal antibodies and cytofluorometric analyses were used for these determinations. There were no significant differences (P = 0.01) between these groups for T-cell markers. A statistically significant (P = 0.001) increased ratio of cells bearing surface immunoglobulin to those expressing Ia antigen (K&L/Ia) was observed between normal non-pregnant controls and women with a history of chronic spontaneous abortion. It is concluded that T-lymphocytes in the peripheral blood do not demonstrate a phenotypic abnormality that would account for the non-rejection of the fetal allograft; however, women with chronic spontaneous abortion may have abnormal B-cell differentiation or T-cell activation that mediates chronic spontaneous abortion.

The cost-effectiveness of routine histologic examination.

Although the histologic examination of routine tissues, such as hernia sacs and intervertebral disks, has shown a low incidence of detecting clinically significant unsuspected disease, the cost-effectiveness of histologic examination has not been determined. By using a theoretical model that assumed variable costs and gains in life expectancy secondary to detecting clinically significant disease, a threshold incidence of disease detection at which histologic examination is cost-effective was determined. By using the University of lowa (Iowa City) cost of examination (approximately $25), at least 1 of every 2,000 examinations would have to show clinically significant disease for histologic examination to be cost-effective. This threshold incidence decreases as production costs decrease or life-year values increase. Before definitive policy conclusions can be made, additional studies are needed to better define the trade-off between cost and the value of information and the incidence of detecting clinically significant disease.

The cost-effectiveness of cervical-vaginal rescreening.

Although most laboratories practice 10% manual rescreening, the cost-effectiveness of this and other rescreening strategies rarely has been evaluated. Using data obtained from the medical literature, a decision model was created in which rescreening strategies were compared with nonrescreening strategies for the number of false-negative and false-positive diagnoses, cancers, life expectancy, and cost-effectiveness. The strategy of 10% rescreening with a repeated cervical-vaginal smear yielded almost no gain in life expectancy compared with an equivalent strategy with no rescreening. With 100% rescreening, the gain in life expectancy was only 0.24 days per patient. A 100% rescreening strategy generally was more cost-effective than a no-rescreening strategy at costs of rescreening varying from $2 to $10 per patient. A 10% rescreening strategy has limited utility. In addition, 100% rescreening strategies are more cost-effective than nonrescreening strategies, but only if the rescreening cost is low.

Fine-needle aspiration biopsy of pulmonary coccidiodomycosis. Spectrum of cytologic findings in 73 patients.

Coccidioides immitis, the causal agent of coccidiodomycosis, is endemic in the arid desert regions of Mexico and the southwestern United States. Individuals may acquire the disease through the inhalation of conidia. The majority of infected patients are asymptomatic or exhibit flu-like symptoms. Two percent of infected individuals ultimately demonstrate a solitary pulmonary nodule that may be radiographically indistinguishable from neoplasms or other infectious lesions. This report describes the spectrum of cytologic findings in 73 patients who were diagnosed with pulmonary coccidiodomycosis by fine-needle aspiration (FNA) biopsy. The patients ranged in age from 30 to 92 years. Ten had a previous history of malignancy. The smears were characterized by a large amount of granular, eosinophilic debris with a paucity of acute or chronic inflammation. Granulomatous inflammation was present in only three cases. The diagnosis was confirmed by the cytologic observation of C. immitis spherules that ranged in size from approximately 20 to 200 microns. Many of these spherules had a crushed or fractured appearance, and occasional calcified forms were seen. Endospores were observed in intact spherules and were rarely observed outside these spherules. Mycelial elements occasionally were present. C. immitis were cultured in 9 of 44 cases in which fungal cultures were obtained. The cytologic differential diagnosis, which includes contaminant, other infectious diseases and malignancies, is discussed.

The cytologic diagnosis of low-grade transitional cell carcinoma.

Current theory suggests that transitional cell carcinoma (TCC) occurs as either of 2 disease processes, each of which has a distinct cytologic appearance and clinical course: low-grade and high-grade TCC. Urinary cytology has become a mainstay technique for monitoring disease recurrence in patients with TCC. Most cases of high-grade TCC can be diagnosed accurately in urinary cytology specimens. However, the cytologic diagnosis of low-grade TCC is difficult; these tumors exhibit subtle cytomorphologic alterations that are difficult to distinguish from benign or reactive processes. The cytologic criteria most useful for diagnosing low-grade TCC in urinary cytology specimens are reviewed. Additionally, the discussion includes some of the new ancillary tests that are emerging as possible diagnostic aids for the detection of low-grade urothelial neoplasms.

The usefulness of pathologists' assistants.

The usefulness of pathologists' assistants (PAs) has not been assessed rigorously. Data from a time-motion self-report log generated by an Allegheny General Hospital (Pittsburgh, PA) PA and from the corresponding surgical specimen logs were reviewed to determine the daily distribution of PA time and multiple parameters of practice for gross examination of specimens. Using these data in specific scenarios, PA and non-PA practices were compared. The majority of the PA's time (56.5%) was spent performing gross examination of surgical biopsy specimens. The average cost of gross examination per specimen for a PA and a pathologist was $4.37 and $15.19, respectively. In this practice setting, $91,970.00 is saved per year by the use of a PA. The use of PAs instead of pathologists results in considerable practice cost savings ($560,000 in a practice of 50,000 specimens) or saves pathologists time to perform other necessary functions. PAs are highly useful in an era of cost containment.

The cost-effectiveness of the cytology laboratory and new cytology technologies in cervical cancer prevention.

The effect of changes in cytology laboratory costs, including the costs of new technologies, on the cost-effectiveness of cervical cancer prevention has not been studied. Using University of Iowa laboratory detection rates and costs, a decision model determined the cost-effectiveness of the laboratory with and without new technologies. Compared with not performing a cervicovaginal smear, the cost to increase the discounted life expectancy per patient by 1 year was $2,805 for the laboratory component alone and $19,655 for the entire cervical cancer prevention strategy. In moderate- to high-risk women, cervical cancer screening was cost-effective even at high cytology laboratory costs (eg, $75 per smear). New technologies were cost-effective only if they resulted in a substantial increase in the detection of high-grade squamous intraepithelial lesions (eg, an additional 236 high-grade squamous intraepithelial lesions per 10,000 women). New technologies have not demonstrated these increased detection rates.

Cost effectiveness of rescreening cervicovaginal smears.

Although cytology laboratories are mandated to rescreen at least 10% of cervicovaginal smears, there is no uniform national rescreening practice. Follow-up data for 16,188 rescreened cervicovaginal smears were studied and decision analysis was performed to determine an optimal rescreening strategy. High-grade dysplasia was detected in 0.40% of women with a history of cervical disease and in 0.04% without a history of cervical disease. Compared with 0% rescreening of smears, with 15% rescreening the cost to gain a year of discounted life expectancy was $386,890 for women without a history of cervical disease, and $2,980 for women with a history of cervical disease. We conclude that rescreening only smears from women with a history of cervical disease could save US laboratories more than $11.2 million annually without seriously compromising care.

Long-term outcome and relative risk in women with atypical squamous cells of undetermined significance.

Few studies have compared long-term follow-up and risk for invasive cancer in women with atypical squamous cells of undetermined significance (ASCUS). We conducted a 6-year review of pathology files for 651 women in whom ASCUS had been diagnosed in 1992. Data collected included patient demographics, follow-up diagnoses, time between follow-up examinations, and procedures performed. At follow-up, high-grade squamous intraepithelial lesions (HSIL) had developed in 9.0% of the women, and invasive cancer in none. Previous cervical history did not affect risk for an HSIL. Although the average time to first follow-up was 6.18 months, in 20.9% of the women the diagnosis of HSIL was not established until after 2.0 years. For individual pathologists, the percentage of HSILs ranged from 0% to 18.8%. Thus women with ASCUS who are followed up regularly are at low risk for development of invasive cancer.