Cell-free DNA analysis in current cancer clinical trials: a review.

Cell-free DNA (cfDNA) analysis represents a promising method for the diagnosis, treatment selection and clinical follow-up of cancer patients. Although its general methodological feasibility and usefulness has been demonstrated, several issues related to standardisation and technical validation must be addressed for its routine clinical application in cancer. In this regard, most cfDNA clinical applications are still limited to clinical trials, proving its value in several settings. In this paper, we review the current clinical trials involving cfDNA/ctDNA analysis and highlight those where it has been useful for patient stratification, treatment follow-up or development of novel approaches for early diagnosis. Our query included clinical trials, including the terms 'cfDNA', 'ctDNA', 'liquid biopsy' AND 'cancer OR neoplasm' in the FDA and EMA public databases. We identified 1370 clinical trials (FDA = 1129, EMA = 241) involving liquid-biopsy analysis in cancer. These clinical trials show promising results for the early detection of cancer and confirm cfDNA as a tool for real-time monitoring of acquired therapy resistance, accurate disease-progression surveillance and improvement of treatment, situations that result in a better quality of life and extended overall survival for cancer patients.

An update on interstitial lung disease.

Interstitial lung diseases are a complex group of conditions that cause inflammation and scarring of the lung interstitium. This article discusses the diagnosis and management of common interstitial lung diseases including idiopathic pulmonary fibrosis, hypersensitivity pneumonitis, connective tissue disease associated-interstitial lung disease, sarcoidosis and drug-induced interstitial lung disease. A multidisciplinary approach to diagnosis of interstitial lung disease is the gold standard; key history and examination features, blood panel, pulmonary function tests, high resolution computed tomography imaging, and when required bronchoalveolar lavage and lung biopsy results are discussed to reach a multidisciplinary consensus diagnosis. Advances, including the development of the disease-modifying anti-fibrotic medications nintedanib and pirfenidone, continue to shape the future management of interstitial lung disease. A holistic approach to the care of patients with interstitial lung disease is paramount, as they often have a high symptom burden and considerable palliative care needs.

European recommendations and quality assurance for cytogenomic analysis of haematological neoplasms.

Cytogenomic investigations of haematological neoplasms, including chromosome banding analysis, fluorescence in situ hybridisation (FISH) and microarray analyses have become increasingly important in the clinical management of patients with haematological neoplasms. The widespread implementation of these techniques in genetic diagnostics has highlighted the need for guidance on the essential criteria to follow when providing cytogenomic testing, regardless of choice of methodology. These recommendations provide an updated, practical and easily available document that will assist laboratories in the choice of testing and methodology enabling them to operate within acceptable standards and maintain a quality service.

Prevalence of psychotropic medication use and association with challenging behaviour in adults with an intellectual disability. A total population study.

BACKGROUND: There is a high prevalence of psychotropic medication use in adults with Intellectual Disabilities (ID), often in the absence of psychiatric disorder, also associated with challenging behaviour. Previous research has focused on specific sample frames or data from primary care providers. There is also a lack of consistency in the definition of challenging behaviour used. METHODS: We adopted a total population sampling method. Medication data on 265 adults with ID were classified according to the Anatomical Therapeutic Chemical classification system. The Behaviour Problems Inventory - short form classified challenging behaviours. We examined the association between challenging behaviour and the use of psychotropic medication, and whether any association would still be present after accounting for socio-demographic and clinical characteristics. RESULTS: 70.57% of adults with ID were prescribed at least one class of any medication (mean per person =2.62; range 0-14). Psychotropic medications were used by 37.73% of participants with antipsychotics the commonest type used by 21.89% of individuals. Polypharmacy and high dosages were common. Generalised Linear Models indicated significant associations between psychotropic medication and the presence of a psychiatric diagnosis, challenging behaviour, older age and type of residence. Male gender was additionally associated with antipsychotic medication. CONCLUSIONS: The use of a total population sample identified via multiple routes is less likely to overestimate prevalence rates of medication use. Current challenging behaviour was a predictor of medication use after controlling for other variables. Data indicate that there may be differences in prescribing patterns associated with different topographies of challenging behaviours.

Cytogenetic Nomenclature: Changes in the ISCN 2013 Compared to the 2009 Edition.

The latest edition of the International System for Human Cytogenetic Nomenclature, ISCN 2013, has recently been published following a thorough revision of the 2009 issue and the incorporation of suggestions from the community by the current standing committee. This review will highlight the multiple nomenclature changes in the respective chapters of the 2013 version compared to the previous version of the ISCN published in 2009. These highlights are meant as a guide for the cytogeneticist to assist in the transition in the use of this updated nomenclature for describing cytogenetic and molecular cytogenetic findings in both clinical and research reports.

Medical, surgical, and health outcomes of gastrostomy feeding.

A prospective controlled study with repeated measures before and after surgery examined the medical, surgical, and health outcomes of gastrostomy for children with disabilities at a tertiary paediatric referral centre in the North Thames area, UK. Anthropometric measures included weight, mid-upper-arm and head circumference. Five-day prospective food diaries were completed and data on physical health and surgical outcomes recorded. Seventy-six children participated and underwent gastrostomy (44 males, 32 females; median age 3 y 4 mo, range 4 mo-17 y 5 mo), and 35/76 required an anti-reflux procedure. Categories of disability were: cerebral palsy (32/76), syndrome of chromosomal or other genetic origin (25/76), slowly progressive degenerative disease (11/76), and unconfirmed diagnosis (8/76). Most children had gross motor difficulties (99%) and were non-ambulant (83%). Oromotor problems were identified in 78% of children, 69% aspirated, and 65% were fed nasogastrically before surgery. The mean weight before surgery was -2.84 standard deviation score (SDS; SD 2.21, range -9.8 to 3.4). Two-thirds of children achieved catch-up growth postoperatively: weight-for-age (mean difference 0.51 SDS, 95% CI 0.23-0.79, p=0.001) and mid-upper arm circumference (mean difference 1.12 cm, 95% confidence interval 0.50-1.75, p=0.001). Health gains included a reduction in drooling, secretions, vomiting, and constipation. Major surgical complications were found in 13/74 children. The study provides evidence that catch-up growth and health gains are possible following gastrostomy.

Monitoring of skeletal progression of prostate cancer by GFP imaging, X-ray, and serum OPG and PTHrP.

BACKGROUND: Prostate cancers (PCas) produce factors that can serve as biomarkers for tumor metastasis and bone progression. Transduced GFP expression by cancer cells can be imaged to monitor therapy. We exploited both concepts by developing a GFP-expressing PCa cell line that expresses PTHrP and studying it in an animal model of malignancy with methods that assess the skeletal progression of this tumor. METHODS: We developed a GFP-producing PCa cell line by stable transduction of PC-3 PCa cells. This PC-3 variant was used to study tumor progression in an immunocompromised mouse model. Skeletal progression of the PCa cells and the effects of pamidronate administration were evaluated radiologically, fluorometrically, and by measurement of serum tumor markers. RESULTS: The PC-3 cells produced extensive bone lesions when injected into the tibia of immunocompromised mice. The skeletal progression of the PC-3 cells could be monitored by GFP optical imaging, X-ray, and by measurements of tumor products in serum, notably PTHrP and OPG. Pamidronate treatment reduced tumor burden as assessed at autopsy by imaging and biomarkers. CONCLUSIONS: Pamidronate treatment exhibited anti-tumor effects that were reflected by decreases in serum PTHrP, OPG, and by GFP and radiological imaging procedures. Imaging of GFP expression enables real-time monitoring of tumor growth in the bone. PTHrP and OPG may be useful as tumor biomarkers for PCa that has metastasized to bone. This novel human PCa model can be used to study the clinical potential of diagnostic and therapeutic modalities in the skeletal progression of PCas.

Coherent expression chromosome cluster analysis reveals differential regulatory functions of amino-terminal and distal parathyroid hormone-related protein domains in prostate carcinoma.

Parathyroid hormone-related protein (PTHrP) has a number of cancer-related actions. While best known for causing hypercalcemia of malignancy, it also has effects on cancer cell growth, apoptosis, and angiogenesis. Studying the actions of PTHrP in human cancer is complicated because there are three isoforms and many derived peptides. Several peptides are biologically active at known or presumed cell surface receptors; in addition, the PTHrP-derived molecules can exert effects at the cell nucleus. To address this complexity, we studied gene expression in a DU 145 prostate cancer cell line that was stably transfected with control vector, PTHrP 1-173 and PTHrP 33-173. With this model, regulatory effects of the amino-terminal portion of PTHrP would result only from transduction with the full-length molecule, while effects pertaining to distal sequences would be evident with either construct. Analysis of the expression profiles by microarrays demonstrated nonoverlapping groups of differentially expressed genes. Amino-terminal PTHrP affected groups of genes involved in apoptosis, prostaglandin and sex steroid metabolism, cell-matrix interactions, and cell differentiation, while PTHrP 33-173 caused substantial increases in MHC class I antigen expression. This work demonstrates the distinct biological actions of the amino-terminus compared to distal mid-molecule or carboxy-terminal sequences of PTHrP in prostate carcinoma cells and provides targets for further study of the malignant process.

Parathyroid hormone-related protein regulates the growth of orthotopic human lung tumors in athymic mice.

BACKGROUND: Parathyroid hormone-related protein (PTHrP) has growth regulatory effects for many malignant cells and may influence the progression of carcinomas of the breast, prostate, and lung. In the current study, the authors investigated the in vivo and in vitro effects of PTHrP neutralizing antibody and PTHrP treatment on the growth of BEN cells, a human lung squamous cell carcinoma line that expresses PTHrP and its receptor. METHODS: Orthotopic lung tumors were produced in 20 athymic mice with BEN-GFP cells (a clonal line that stably expresses green fluorescent protein [GFP]) by instilling suspensions of 3 x 10(6) cells per mouse into the lungs of anesthetized animals. The mice were divided into 2 groups receiving either subcutaneous mouse antihuman PTHrP antibodies or irrelevant mouse immunoglobulin (Ig) G (150 microg) twice weekly. RESULTS: After 30 days, 6 of 10 mice receiving anti-PTHrP antibodies had lung tumors visible on macroscopic inspection, but only 1 of the 10 mice treated with irrelevant IgG had a lung tumor that was of that size (P < 0.01). GFP fluorescence was significantly greater in lung homogenates of the PTHrP antibody-treated mice than in the mice treated with IgG (6006 +/- 411 vs. 2907 +/- 282 relative fluorescent units, respectively; P < 0.001). Although neutralizing antibodies stimulated BEN cell lung tumor growth, exogenous PTHrP 1-34 treatment (0.01-1 nM) inhibited the growth of cultured BEN cells by approximately 40%. CONCLUSIONS: Although PTHrP expression has been reported to be associated with more aggressive malignancies, the data from the current study suggest that PTHrP 1-34 was a paracrine growth inhibitor in BEN human lung carcinoma cells. The growth-related effects of PTHrP are complex, and can be both stimulatory and inhibitory.

Comparative tissue distribution of the processing enzymes "prohormone thiol protease," and prohormone convertases 1 and 2, in human PTHrP-producing cell lines and mammalian neuroendocrine tissues.

Peptide hormones are generated by proteolytic processing of their respective protein precursors by several prohormone processing proteases. The peptide hormone PTHrP is widely expressed in normal and malignant tissues, where proPTHrP undergoes proteolytic processing to generate PTHrP peptides with distinct biological actions. In this study, the tissue distribution of the prohormone processing enzymes PTP, PC1, and PC2 were compared by immunohistochemistry in human PTHrP-producing cancer cell lines, and in mammalian neuroendocrine and other tissues from rat and bovine that contain peptide hormones. PTP, PC1, and PC2 were prominently expressed in PTHrP-expressing human cancer cell lines originating from tumors of the breast, lung, prostate, as well as lymphoma. These processing enzymes also showed significant expression in normal mammalian neuroendocrine tissues from bovine and rat, including pituitary, hypothalamus, adrenal medulla, pancreas, and other tissues. Most neuroendocrine tissues contained prominent levels of at least two of the three processing enzymes examined, and all tissues contained at least one of these three enzymes. Differential expression of processing enzyme proteins was also demonstrated by Western blots. The differential expression of PTP, PC1, and PC2 observed in certain cancer and normal neuroendocrine cell types postulates selective roles for these processing enzymes in different tissues for generating biologically active peptide hormones. These results support the importance of these processing enzymes in their hypothesized roles in prohormone processing.

Parathyroid hormone-related protein and lung injury after pulmonary thromboendarterectomy.

Parathyroid hormone-related protein (PTHrP) is an autocrine growth and differentiation factor for alveolar type II epithelial cells. Type II cells secrete pulmonary surfactant and are pluripotent cells with a role in alveolar epithelial repair after lung injury. The goals of this study were to investigate whether the levels of PTHrP in bronchoalveolar lavage liquid (BAL) varied between patients who did and did not develop lung injury after pulmonary thromboendarterectomy (PTE). BAL was performed in 48 patients undergoing PTE for unresolved pulmonary emboli. Samples were obtained following induction of anesthesia, following separation from cardiopulmonary bypass, and 48 h postoperatively. PTHrP was measured by radioimmunoassay. Lung injury was diagnosed in 23 patients on the basis of hypoxemia (PaO(2)/FIO(2) < 300) and the presence of lung infiltrates in the absence of infection or atelectasis. Patients with lung injury had significantly lower preoperative BAL levels of PTHrP, 21 (21-30) pg/ml (median, interquartile gap), compared to patients without lung injury, 34 (21-41) pg/ml (P < 0.05). Preoperative BAL PTHrP levels < 32 pg/ml predicted lung injury with a positive predictive value of 60% and negative predictive value of 82%. The odds of developing lung injury for patients with preoperative PTHrP levels below this cutpoint were 6 times the odds for patients with higher levels.

Proteolysis of ProPTHrP(1-141) by "prohormone thiol protease" at multibasic residues generates PTHrP-related peptides: implications for PTHrP peptide production in lung cancer cells.

The parathyroid hormone-related protein (PTHrP) precursor requires proteolytic processing to generate PTHrP-related peptide products that possess regulatory functions in the control of PTH-like (parathyroid-like) actions and cell growth, calcium transport, and osteoclast activity. Biologically active peptide domains within the PTHrP precursor are typically flanked at their NH2- and COOH-termini by basic residue cleavage sites consisting of multibasic, dibasic, and monobasic residues. These basic residues are predicted to serve as proteolytic cleavage sites for converting the PTHrP precursor into active peptide products. The coexpression of the prohormone processing enzyme PTP ("prohormone thiol protease") in PTHrP-containing lung cancer cells, and the lack of PTP in cell lines that contain little PTHrP, implicate PTP as a candidate processing enzyme for proPTHrP. Therefore, in this study, PTP cleavage of recombinant proPTHrP(1-141) precursor was evaluated by MALDI mass spectrometry to identify peptide products and cleavage sites. PTP cleaved the PTHrP precursor at the predicted basic residue cleavage sites to generate biologically active PTHrP-related peptides that correspond to the NH2-terminal domain (residues 1-37) that possesses PTH-like and growth regulatory activities, the mid-region domain (residues 38-93) that regulates calcium transport, and the COOH-terminal domain (residues 102-141) that modulates osteoclast activity. Lack of cleavage at other types of amino acids demonstrated the specificity of PTP processing at basic residue cleavage sites. Overall, these results demonstrate the ability of PTP to cleave the PTHrP precursor at multibasic, dibasic, and monobasic residue cleavage sites to generate active PTHrP-related peptides. The presence of PTP immunoreactivity in PTHrP-containing lung cancer cells suggests PTP as a candidate processing enzyme for the PTHrP precursor.

Aneuploidy screening in direct chorionic villus samples by fluorescence in situ hybridisation: the use of commercial probes in a clinical setting.

The results of screening for the common aneuploidies involving chromosomes 13, 18, 21, X and Y by florescent in-situ hybridisation (FISH) in direct preparations from 100 chorionic villus samples from pregnancies between 10 and 20 weeks' gestation are reported. Samples prepared using routine methods and analysed with commercially available probes, accurately detected 12 cases of fetal aneuploidy, all referred because of developmental abnormality. Three of the four cases where chromosome abnormality was detected in cultured villi but not by the direct fluorescence in situ hybridisation (FISH) assay, were due to confined placental mosaicism. No chromosomal anomalies were found in the 20 low risk cases where the referral reason was a familial single gene disorder. We conclude that the FISH assay with commercial probes may act as an accurate and less labour intensive alternative to direct chromosome analysis of chorionic villus samples. In cytogenetically low risk cases its use can obtain a result within the time needed for DNA analysis and avoid the need to set up cultures.

Splenic, thymic, bony and lymph node metastases from orthotopic human lung carcinomas in immunocompromised mice.

In orthotopic animal models of human lung cancer, bone and lymph node metastases have been observed with high frequency after periods of a few weeks, but metastases to other organs are rare. This study evaluated development of distant metastases over a six-month period in a model of orthotopic lung carcinoma in immunocompromised mice. Human A549 lung adenocarcinoma cells were stably transfected to express high levels of green fluorescent protein. Suspensions of 1 x 10(6) cells were instilled into the lungs of athymic and SCID mice to produce orthotopic human lung carcinomas. All animals had primary tumors at termination of the experiment six months later. Splenic metastases and lymph node metastases were present in 70% of the animals and two of the three SCID mice had thymic metastases. Three animals had bony metastases. Thus, a high percentage of immunocompromised mice with orthotopic lung carcinomas ultimately develop metastases.

Parathyroid hormone-related protein reduces alveolar epithelial cell proliferation during lung injury in rats.

Parathyroid hormone-related protein (PTHrP) is a growth inhibitor for alveolar type II cells and could be a regulatory factor for alveolar epithelial cell proliferation after lung injury. We investigated lung PTHrP expression in rats exposed to 85% oxygen. Lung levels of PTHrP were significantly decreased between 4 and 8 days of hyperoxia, concurrent with increased expression of proliferating cell nuclear antigen and increased incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA in lung corner cells. PTHrP receptor was present in both normal and hyperoxic lung. To test whether the fall in PTHrP was related to cell proliferation, we instilled PTHrP into lungs on the fourth day of hyperoxia. Eight hours later, BrdU labeling in alveolar corner cells was 3.2 +/- 0.4 cells/high-power field in hyperoxic PBS-instilled rats compared with 0.5 +/- 0.3 cells/high-power field in PTHrP-instilled rats (P < 0. 01). Thus PTHrP expression changes in response to lung injury due to 85% oxygen and may regulate cell proliferation.

Localisation of 26S proteasomes with different subunit composition in insect muscles undergoing programmed cell death.

The 26S proteasome is a large multisubunit complex involved in degrading both cytoplasmic and nuclear proteins. We have investigated the subcellular distribution of four regulatory ATPase subunits (S6 (TBP7/MS73), S6' (TBP1), S7 (MSS1), and S10b (SUG2)) together with components of 20S proteasomes in the intersegmental muscles (ISM) of Manduca sexta during developmentally programmed cell death (PCD). Immunogold electron microscopy shows that S6 is located in the heterochromatic part of nuclei of ISM fibres. S6' is present in degraded material only outside intact fibres. S7 can be detected in nuclei, cytoplasm and also in degraded material. S10b, on the other hand, is initially found in nuclei and subsequently in degraded cytoplasmic locations during PCD. 20S proteasomes are present in all areas where ATPase subunits are detected, consistent with the presence of intact 26S proteasomes. These results are discussed in terms of heterogeneity of 26S proteasomes, 26S proteasome disassembly and the possible role of ATPases in non-proteasome complexes in the process of PCD. Cell Death and Differentiation (2000) 7, 1210 - 1217.