Small-Molecule Targeting of BET Proteins in Cancer.

BET proteins have recently become recognized for their role in a broad range of cancers and are defined by the presence of two acetyl-histone reading bromodomains and an ET domain. This family of proteins includes BRD2, BRD3, BRD4, and BRDT. BRD4 is the most-studied BET protein in cancer, and normally serves as an epigenetic reader that links active chromatin marks to transcriptional elongation through activation of RNA polymerase II. The role of BRD3 and BRD4 first became known in cancer as mutant oncoproteins fused to the p300-recruiting NUT protein in a rare aggressive subtype of squamous cell cancer known as NUT midline carcinoma (NMC). BET inhibitors are acetyl-histone mimetics that specifically bind BET bromodomains, competitively inhibiting its engagement with chromatin. The antineoplastic effects of BET inhibitors were first demonstrated in NMC and have since been shown to be effective at inhibiting the growth of many different cancers, particularly acute leukemia. BET inhibitors have also been instrumental as tool compounds that have demonstrated the key role of BRD4 in driving NMC and non-NMC cancer growth. Many clinical trials enrolling patients with hematologic and solid tumors are ongoing, with encouraging preliminary findings. BET proteins BRD2, BRD3, and BRD4 are expressed in nearly all cells of the body, so there are concerns of toxicity with BET inhibitors, as well as the development of resistance. Toxicity and resistance may be overcome by combining BET inhibitors with other targeted inhibitors, or through the use of novel BET inhibitor derivatives.

NUT Carcinoma of the Sublingual Gland.

NUT carcinoma (NC) is a recently described, rare and extremely aggressive cancer primarily located to supradiaphragmatic structures and affecting young individuals. NC is characterized by translocations involving the NUT gene on 15q14 with the most common translocation partner gene being BRD4 on 19p13, resulting in the t(15;19)(q14;p13) karyotype. NC is poorly differentiated and is likely to be overlooked and misdiagnosed as poorly differentiated squamous cell carcinoma (SCC) when immunohistochemical evaluation of NUT protein expression is omitted. Previously, NC has been found in the parotid and submandibular glands and we present the first case in the sublingual gland arising in a 40-year-old woman. We discuss the diagnostic considerations for poorly differentiated carcinomas of the salivary glands and advocate the inclusion of NUT immunohistochemistry in this setting. Not only does the NC diagnosis confer a grave prognosis when treated as SCC as illustrated by the present case, but is important for the inclusion of patients in ongoing clinical trials.

An aetiological Foxp2 mutation causes aberrant striatal activity and alters plasticity during skill learning.

Mutations in the human FOXP2 gene cause impaired speech development and linguistic deficits, which have been best characterised in a large pedigree called the KE family. The encoded protein is highly conserved in many vertebrates and is expressed in homologous brain regions required for sensorimotor integration and motor-skill learning, in particular corticostriatal circuits. Independent studies in multiple species suggest that the striatum is a key site of FOXP2 action. Here, we used in vivo recordings in awake-behaving mice to investigate the effects of the KE-family mutation on the function of striatal circuits during motor-skill learning. We uncovered abnormally high ongoing striatal activity in mice carrying an identical mutation to that of the KE family. Furthermore, there were dramatic alterations in striatal plasticity during the acquisition of a motor skill, with most neurons in mutants showing negative modulation of firing rate, starkly contrasting with the predominantly positive modulation seen in control animals. We also observed striking changes in the temporal coordination of striatal firing during motor-skill learning in mutants. Our results indicate that FOXP2 is critical for the function of striatal circuits in vivo, which are important not only for speech but also for other striatal-dependent skills.

BRD-NUT oncoproteins: a family of closely related nuclear proteins that block epithelial differentiation and maintain the growth of carcinoma cells.

An unusual group of carcinomas, here termed nuclear protein in testis (NUT) midline carcinomas (NMC), are characterized by translocations that involve NUT, a novel gene on chromosome 15. In about 2/3rds of cases, NUT is fused to BRD4 on chromosome 19. Using a candidate gene approach, we identified two NMCs harboring novel rearrangements that result in the fusion of NUT to BRD3 on chromosome 9. The BRD3-NUT fusion gene encodes a protein composed of two tandem chromatin-binding bromodomains, an extra-terminal domain, a bipartite nuclear localization sequence, and almost the entirety of NUT that is highly homologous to BRD4-NUT. The function of NUT is unknown, but here we show that NUT contains nuclear localization and export sequences that promote nuclear-cytoplasmic shuttling via a leptomycin-sensitive pathway. In contrast, BRD3-NUT and BRD4-NUT are strictly nuclear, implying that the BRD moiety retains NUT in the nucleus via interactions with chromatin. Consistent with this idea, FRAP studies show that BRD4, BRD4-NUT and BRD3-NUT have significantly slower rates of lateral nuclear diffusion than that of NUT. To investigate the functional role of BRD-NUT fusion proteins in NMCs, we investigated the effects of siRNA-induced BRD3-NUT and BRD4-NUT withdrawal. Silencing of these proteins in NMC cell lines resulted in squamous differentiation and cell cycle arrest. Together, these data suggest that BRD-NUT fusion proteins contribute to carcinogenesis by associating with chromatin and interfering with epithelial differentiation.

BRD4 bromodomain gene rearrangement in aggressive carcinoma with translocation t(15;19).

Translocation t(15;19)(q13;p13.1) defines a lethal midline carcinoma arising adjacent to respiratory tract in young people. To characterize molecular alterations responsible for the distinctly aggressive biological behavior of this cancer, we mapped the chromosome 15 and 19 translocation breakpoints by fluorescence in situ hybridization (FISH) and Southern blotting. To evaluate preliminarily the frequency, anatomical distribution, and histological features of t(15;19) cancer, we developed a FISH assay for paraffin sections. Our findings reveal a novel oncogenic mechanism in which the chromosome 19 translocation breakpoint interrupts the coding sequence of a bromodomain gene, BRD4. These studies implicate BRD4 as a potential partner in a t(15;19)-associated fusion oncogene. In addition, we localized the chromosome 15 breakpoint to a 9-kb region in each of two cases, thereby identifying several candidate oncogenes which might represent the BRD4 fusion partner. FISH evaluation of 13 pediatric carcinomas revealed t(15;19) in one of four sinonasal carcinomas, whereas this translocation was not detected in thymic (n = 3), mucoepidermoid (n = 3), laryngeal (n = 2), or nasopharyngeal (n = 1) carcinomas. Our studies shed light on the oncogenic mechanism underlying t(15;19) and provide further evidence that this highly lethal cancer arises from respiratory mucosa.

Upper respiratory tract carcinoma with chromosomal translocation 15;19: evidence for a distinct disease entity of young patients with a rapidly fatal course.

BACKGROUND: Carcinoma of the upper respiratory tract is rare in childhood, and cytogenetic aberrations have not been characterized in this population. The chromosomal translocation 15;19 has been reported four times previously. All patients were young and had tumors arising in the thorax. The three reports that provide clinical follow-up all describe superior vena cava syndrome and death soon after presentation. All tumors were diagnosed as carcinoma (three undifferentiated, one mucoepidermoid), and the authors suggested thymus, lung, or germ cell origin. METHODS: The authors investigated the clinical and pathologic findings in two patients with poorly differentiated carcinoma showing evidence of t(15;19). This included a 13-year-old girl with a rapidly growing epiglottic mass, leading to superior vena cava syndrome and death and a 12-year-old girl with an aggressive nasopharyngeal mass showing intracranial extension. RESULTS: The laryngeal tumor was poorly differentiated, with vesicular nuclei, prominent nucleoli, extensive necrosis, and a lymphoplasmacytic infiltrate; cells were positive for cytokeratin and negative for lymphoma, melanoma, germ cell, and endocrine markers. Electron microscopy showed rare intermediate junctions and basal lamina. The nasopharyngeal tumor was poorly differentiated with areas of obvious squamous differentiation observed histologically, immunophenotypically, and ultrastructurally. Cytogenetic and fluorescent in situ hybridization studies were consistent with t(15;19)(q13;p13.1) in both cases. Both children received chemo- and radiotherapy. The first child died of disease after 36 weeks; autopsy revealed tumor in the larynx with spread to the skin/subcutis (neck and thorax) and lymph nodes (cervical, subcarinal, and pulmonary hilar). The second child developed widespread bony metastases and died of disease after 13 weeks. CONCLUSIONS: In conjunction with previous reports, the authors' findings show that t(15;19) is part of a distinct clinicopathologic entity characterized by young age, midline carcinoma of the neck or upper thorax, and a rapidly fatal course. Female gender and superior vena cava syndrome are common. The histogenesis of these distinctive tumors is unknown. The authors' findings suggest origin in the upper airway, perhaps from submucosal glands.

Diagnosing lymphoproliferative disorders involving the cerebrospinal fluid: increased sensitivity using flow cytometric analysis.

Flow cytometric immunophenotypic analysis (FCA) can be performed to evaluate lymphoid cells in cerebrospinal fluid (CSF). We compared this method with conventional cytologic diagnosis to determine its utility. A retrospective comparison of 35 consecutive CSF flow cytometry results with the corresponding cytologic diagnoses was undertaken. Twenty-five of 35 CSFs (71%) were successfully analyzed by flow cytometry. The 10 samples which could not be analyzed were either too old (greater than 3 days) or had an insufficient number of cells. A total of 9 lymphomas was detected: 4 by both flow cytometry and cytology; 2 by cytology alone; and 3 by flow cytometry alone. This represents a 50% increase in the detection of lymphoproliferative disorders in CSF by a combination of flow cytometry and cytology vs. cytology alone. Furthermore, in 3 cases with follow-up where the cytologic diagnosis was "atypical cells of undetermined significance" and the flow cytometric findings were negative for malignancy, the clinical course confirmed a benign pleocytosis in all three. We conclude that flow cytometric analysis markedly improves sensitivity when used in combination with cytology in the evaluation of lymphoid cells in CSF.

Intradermal spindle cell/pleomorphic lipoma: a distinct subset.

Spindle cell/pleomorphic lipomas are a group of benign lipogenic tumors composed of primitive spindle cells, multinucleated giant cells, and mature adipocytes. These tumors have rarely been reported to arise in the dermis and may be misdiagnosed in this location. Twenty (12.7%) intradermal lesions identified among 157 spindle cell/pleomorphic lipomas in the authors' files were studied clinicopathologically and immunohistochemically. The patients' ages ranged from 20 to 85 years (median: 42 years); 14 of 20 patients were female (70%). Anatomical sites were the head/neck region (7 cases, 4 of which arose on the face), shoulder/upper back (4 cases), lower limbs (4 cases), trunk (3 cases), and upper limbs (2 cases). Most lesions presented as a soft and slowly enlarging cutaneous nodule, usually measuring less than 2.5 cm. Histologically, these dermal lesions differed from usual spindle cell/pleomorphic lipoma, being unencapsulated with poorly defined infiltrative margins, although the cytomorphologic findings, ropy collagen, and mast cells were as seen in usual subcutaneous lesions. Six cases showed features of pleomorphic lipoma. Immunohistochemically, lesional cells stained positively for CD34 and were negative for S-100 protein. One case recurred locally after 21 years. Dermal spindle cell/pleomorphic lipomas are distinctive in their apparent female predilection, wider anatomical distribution than subcutaneous lesions, and lack of circumscription. These unusual features can cause problems in differential diagnosis.

Second-trimester maternal serum progesterone levels in Turner syndrome with and without hydrops and in trisomy 18.

Placental proteins, such as inhibin A and hCG and its subunits, as well as the placental steroid progesterone, are elevated in second-trimester maternal serum from cases of fetal Down syndrome. Since different cellular mechanisms are required for protein versus steroid synthesis and secretion, these data suggest that a generalized placental hypersecretory phenomenon is associated with Down syndrome. Inhibin A and hCG are also elevated in cases of Turner syndrome with hydrops, and are reduced in cases of Turner syndrome without hydrops and in trisomy 18. The objective of the present study was to determine maternal serum levels of the placental steroid progesterone in cases of Turner syndrome and trisomy 18. Twenty-one cases of trisomy 18, 10 cases of Turner syndrome without hydrops and 12 cases of Turner syndrome with hydrops were identified and each matched to five control samples. Maternal serum progesterone levels were significantly elevated in Turner syndrome with hydrops (2.11 MoM), slightly reduced in Turner syndrome without hydrops (0.90 MoM) and modestly, though significantly, reduced in trisomy 18 (0.73 MoM). These data are similar to the patterns seen for inhibin A and hCG, suggesting that the overall synthetic and/or secretory activity of the placenta is increased in Turner syndrome with hydrops and decreased in Turner syndrome without hydrops and in trisomy 18. These data may be helpful in understanding the pathophysiological basis of serum marker patterns in these aneuploidies.

Second-trimester maternal serum inhibin A levels in fetal trisomy 18 and Turner syndrome with and without hydrops.

The objective was to investigate whether cases of fetal trisomy 18 and Turner syndrome with and without hydrops were associated with alterations in the second-trimester levels of maternal serum inhibin A. Twenty-one cases of trisomy 18, 10 cases of Turner syndrome without hydrops and 12 cases of Turner syndrome with hydrops were identified. Five control samples were matched to each case for date of sample collection and completed week of gestation. Inhibin A levels were modestly, but significantly reduced in cases of trisomy 18 (median = 0.88 MoM) and Turner syndrome without hydrops (median = 0.64 MoM). In contrast, inhibin A levels were markedly increased in cases of Turner syndrome with hydrops (median = 3.91 MoM). These data for Turner syndrome are similar to those for human chorionic gonadotropin (hCG). The addition of inhibin A to multiple marker screening (alpha-fetoprotein, unconjugated oestriol and hCG) resulted in a median increase in the Down syndrome risk of 2.6-fold in cases of Turner syndrome with hydrops. The addition of inhibin A to multiple marker Down syndrome screening programmes will be likely to enhance the detection of fetal Turner syndrome with hydrops, but will not contribute substantially to the detection of fetal trisomy 18.

Twins, placentas, and genetics: acardiac twinning in a dichorionic, diamniotic, monozygotic twin gestation.

We describe a human acardiac twin with associated vascular anastomoses in a dichorionic diamniotic fused twin placenta. A 22-year-old woman delivered a healthy 3,554 g male infant and a fused diamniotic dichorionic twin placenta with a 230 g umbilical cord-attached, skin-covered, ovoid mass, consistent with acardiac amorphus. By gross and histological examination, the placental dividing membranes comprised four leaves, one amnion from each placenta, and two centrally fused chorions, diagnostic of dichorionicity. Placental barium injection of the normal twin's umbilical vein showed an anastomosis with the acardiac twin which traversed the dividing membranes, then supplied major vessels of the acardiac mass via its 5.5 cm umbilical cord. DNA-typing studies of the normal twin's placenta and of the acardiac twin's tissues revealed identical alleles at 11 distinct genetic polymorphic loci, consistent with monozygosity. Our findings demonstrate that vascular anastomoses can occur in dichorionic twin placentas, and that human acardiac twinning is not, as heretofore believed, restricted to monochorionic placentas.

Maternal serum analyte levels in pregnancies with fetal Down syndrome resulting from translocations.

OBJECTIVE: Our purpose was to determine whether pregnancies affected by fetal Down syndrome resulting from Robertsonian translocations are associated with second-trimester maternal serum analyte levels different from those resulting from fetal trisomy 21. STUDY DESIGN: Pregnancies with Down syndrome caused by Robertsonian translocations were identified through the cytogenetics laboratories at the participating institutions. Those with maternal serum screening values between 15 and 20 weeks were evaluated. RESULTS: Eleven cases of fetal Down syndrome caused by Robertsonian translocations were identified. The median alpha-fetoprotein, unconjugated estriol, and human chorionic gonadotropin levels were 0.68, 0.67, and 2.83 multiples of the median, respectively. These analyte levels are similar to those for fetal trisomy 21. CONCLUSIONS: These data suggest that Down syndrome resulting from either Robertsonian translocations or trisomy 21 will be detected in a similar percentage of cases because the second-trimester maternal serum analyte levels are similar.

Mechanisms of stabilization of the insulin hexamer through allosteric ligand interactions.

The insulin hexamer is an allosteric protein capable of undergoing transitions between three conformational states: T6, T3R3, and R6. These transitions are mediated by the binding of phenolic compounds to the R-state subunits, which provide positive homotropic effects, and by the coordination of anions to the bound metal ions, which act as heterotropic effectors. Since the insulin monomer is far more susceptible than the hexamer to thermal, mechanical, and chemical degradation, insulin-dependent diabetic patients rely on pharmaceutical preparations of the Zn-insulin hexamer, which act as stable forms of the biologically active monomeric insulin. In this study, the chromophoric chelator 2,2',2"-terpyridine (terpy) has been used as a kinetic probe of insulin hexamer stability to measure the effect of homotropic and heterotropic effectors on the dissociation kinetics of the Zn2+- and Co2+-insulin hexamer complexes. We show that the reaction between terpy and the R-state-bound metal ion is limited by the T3R3 <==> T6 or R6 <==> T3R3 conformational transition steps and the dissociation of one anionic ligand, or one anionic ligand and three phenolic ligand molecules, respectively, for T3R3 and R6. Consequently, because the activation energies of these steps are dominated by the ground-state stabilization energy of the R-state species, the kinetic stabilization of the insulin hexamer toward terpy-induced dissociation is linked to the thermodynamic stabilization of the hexamer. The mass action effect of anion binding and, foremost, of phenolic ligand binding provides the major mechanism of stabilization, resulting in the tightening of the tertiary and quaternary hexamer structures. Using this kinetic method, we show that the R6 conformation of Zn-insulin in the presence of Cl- ion and resorcinol is > 1.5 million-fold more stable than the T3 units of T6 and T3R3 and > 70,000-fold more stable than the R3 unit of T3R3. Furthermore, the stabilization effect is correlated with the affinity of the ligands: the tighter the binding, the slower the reaction between terpy and R-state-bound metal ion. These concepts provide a new basis for the pharmaceutical improvement of the physicochemical stability of formulations both for native insulin and for fast-acting monomeric insulin analogues through ligand-mediated allosteric interactions.

Decreased maternal serum hCG levels with increasing gravidity and parity.

OBJECTIVE: To investigate the preliminary observation that primigravid women have higher hCG multiples of the median (MoM) than multigravid women. METHODS: An analysis of the effect of gravidity and parity on maternal serum alpha-fetoprotein (MSAFP) and hCG was performed using data from 20,009 consecutive singleton pregnancies of 15-20 weeks' gestation in a maternal serum screening program. RESULTS: The human chorionic gonadotropin MoM for primigravid women was 0.1 MoM higher than for multigravid women. As parity or gravidity increased, maternal serum hCG decreased. The median hCG MoM for nulliparous women was 1.05, compared with 0.94 MoM for para 3 women. The decrease in hCG was similar at each gestational week from 15-20. In contrast, MSAFP and MSAFP MoM were unaffected by parity. Maternal age and race were potential contributing factors to the effect of parity. However, the decrease in hCG MoM with parity was observed within each 5-year increment of maternal age. Similarly, both black and non-black populations displayed decreases in hCG with parity, although black women had a consistently higher MoM in all matched sets. The decrease in hCG MoM with parity was also observed in 50 Down syndrome cases. Correcting patient data for parity resulted in the hCG MoM changing only 2.7% on average. The detection rate for the 50 Down syndrome cases would not have changed. CONCLUSION: The decrease in maternal serum hCG with increasing parity demonstrates that pregnancy history influences the level of maternal serum hCG. Further studies are needed to define the contributing factors, but the impact of parity on Down syndrome screening appears to be small.

Effectiveness of combining maternal serum alpha-fetoprotein and hCG in a second-trimester screening program for Down syndrome.

OBJECTIVE: To evaluate the efficacy of combining hCG and alpha-fetoprotein (AFP) with maternal age in a two-analyte maternal serum screening program for Down syndrome. METHODS: A prospective study involved the screening of 12,170 maternal sera from patients at 14-25 weeks of gestation. The risk for Down syndrome at term was calculated from maternal serum hCG and AFP, and maternal age. For women 36 years of age and younger, a risk of 1:307 or greater was considered screen-positive. For women over 36, a risk greater than that a priori was considered screen-positive. False-positive rates and detection rates were compared with those resulting from a screening protocol using only AFP and age. RESULTS: Seven hundred eighty-two sera were initially screen-positive (6.4%). Subsequent sonography decreased this total to 687 (5.6%), and 467 (3.8%) of these patients accepted amniocentesis. Ten cases of Down syndrome and seven other chromosomal abnormalities were detected. Follow-up investigations revealed eight additional Down syndrome cases that were missed by screening. The identification of 18 Down syndrome cases in 12,170 pregnancies corresponds closely with the prediction of 14.1 Down syndrome births (18.2 second-trimester fetuses) in this population calculated from age-dependent risks. The detection rate for Down syndrome was 56% (ten of 18 expected cases). Only five of 18 (28%) would have been detected by AFP and age alone. CONCLUSION: These results support the mathematical model that hCG is the major contributor to the increased sensitivity of multi-analyte screening and demonstrate that screening programs can attain substantial improvement in detection of second-trimester Down syndrome by adding hCG to AFP and age.

Precision and accuracy of a portable blood analyzer system during cholesterol screening.

The precision and accuracy of two Kodak Ektachem DT-60 portable blood analyzers were assessed in a model (research) cholesterol screening program in Rochester, New York. Between June and October 1987, a total of 8,573 people underwent a cholesterol screening held in a movable trailer. A wide variety of temperature, humidity, and other potentially adverse conditions were encountered during the screening period. Between-run coefficients of variation ranged from 1.9 percent to 4.8 percent per month; average bias compared to a Reference Laboratory method ranged between +0.2 percent and +2.0 percent. Both precision and accuracy met currently recommended standards for cholesterol testing in the United States.

Relative diagnostic value of cerebrospinal fluid kappa chains in MS: comparison with other immunoglobulin tests.

We used receiver operating characteristic (ROC) curve analysis to determine the relative performance of different CSF IgG parameters as diagnostic markers for multiple sclerosis. We quantitated CSF and serum IgG and albumin to determine an IgG albumin ratio, IgG index, Tourtellotte's synthesis rate, and Schuller's formula; CSF free kappa light chains were measured by radioimmunoassay. We compared a group of patients with clinically definite MS with a group of patients with a variety of other neurologic diseases. Tourtellotte's formula, IgG albumin ratio, IgG index, and free kappa chains distinguished the MS group from the comparative group of patients. ROC curve analysis demonstrated that CSF free kappa light chains were more closely linked to MS than the other measures. Multiple logistic regression analysis demonstrated no advantage of adding any single IgG measure or combination of measures to free kappa light chain analysis. Our results strongly suggest that free kappa light chains in CSF are the single best quantitative assay to support a clinical diagnosis of MS.

Functionally distinct agretopic and epitopic sites. Analysis of the dominant T cell determinant of moth and pigeon cytochromes c with the use of synthetic peptide antigens.

The dominant T cell determinant on moth and pigeon cytochromes c in B10.A (E beta k:E alpha k) mice is located in the C-terminal portion of the protein, contained within residues 93-103 or 93-104. Thirty-seven antigen analogs, containing single amino acid substitutions at positions 98, 99, 101, 102, 103, and 104, were synthesized. The effects of the substitutions on in vitro antigenicity and in vivo immunogenicity were determined. Functional assays with T cell clones identified residues 99, 101, 102, and 103 as critical, based on their effect on antigenic potency. Peptides containing substitutions at residues 99, 101, and 102 were capable of eliciting unique clones upon immunization of B10.A mice. This was consistent with the identification of these residues as part of the epitope, the site on the antigen that interacts with the T cell receptor. Immunization with peptides substituted at residue 103, however, failed to elicit clones with unique specificity for the immunogen. When these peptides were tested for their ability to stimulate the T cell clones with antigen-presenting cells from B10.A(5R) mice expressing the E beta b:E alpha k Ia molecule, a consistent change in the relative antigenic potency was observed with 50% of the peptides. The effect of the Ia molecule on the antigenic potency ruled out the possibility that residue 103 nonspecifically affected antigen uptake or processing and identified residue 103 as part of the agretope, the site that interacts with the Ia molecule. The locations of the agretope and the epitope on this antigenic determinant appear to be fixed, even in the presence of large numbers of amino acid substitutions. However, some substitutions were found to affect both the agretope and the epitope, placing limits on the functional independence of the two sites. The results are discussed in terms of the trimolecular complex model of T cell activation and the implications of these data for antigen-Ia molecule interactions.