Dynamic changes in the clonal structure of MDS and AML in response to epigenetic therapy.

Traditional response criteria in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are based on bone marrow morphology and may not accurately reflect clonal tumor burden in patients treated with non-cytotoxic chemotherapy. We used next-generation sequencing of serial bone marrow samples to monitor MDS and AML tumor burden during treatment with epigenetic therapy (decitabine and panobinostat). Serial bone marrow samples (and skin as a source of normal DNA) from 25 MDS and AML patients were sequenced (exome or 285 gene panel). We observed that responders, including those in complete remission (CR), can have persistent measurable tumor burden (that is, mutations) for at least 1 year without disease progression. Using an ultrasensitive sequencing approach, we detected extremely rare mutations (equivalent to 1 heterozygous mutant cell in 2000 non-mutant cells) months to years before their expansion at disease relapse. While patients can live with persistent clonal hematopoiesis in a CR or stable disease, ultimately we find evidence that expansion of a rare subclone occurs at relapse or progression. Here we demonstrate that sequencing of serial samples provides an alternative measure of tumor burden in MDS or AML patients and augments traditional response criteria that rely on bone marrow blast percentage.

IRF6 mutation screening in non-syndromic orofacial clefting: analysis of 1521 families.

Van der Woude syndrome (VWS) is an autosomal dominant malformation syndrome characterized by orofacial clefting (OFC) and lower lip pits. The clinical presentation of VWS is variable and can present as an isolated OFC, making it difficult to distinguish VWS cases from individuals with non-syndromic OFCs. About 70% of causal VWS mutations occur in IRF6, a gene that is also associated with non-syndromic OFCs. Screening for IRF6 mutations in apparently non-syndromic cases has been performed in several modestly sized cohorts with mixed results. In this study, we screened 1521 trios with presumed non-syndromic OFCs to determine the frequency of causal IRF6 mutations. We identified seven likely causal IRF6 mutations, although a posteriori review identified two misdiagnosed VWS families based on the presence of lip pits. We found no evidence for association between rare IRF6 polymorphisms and non-syndromic OFCs. We combined our results with other similar studies (totaling 2472 families) and conclude that causal IRF6 mutations are found in 0.24-0.44% of apparently non-syndromic OFC families. We suggest that clinical mutation screening for IRF6 be considered for certain family patterns such as families with mixed types of OFCs and/or autosomal dominant transmission.

Clonal evolution revealed by whole genome sequencing in a case of primary myelofibrosis transformed to secondary acute myeloid leukemia.

Clonal architecture in myeloproliferative neoplasms (MPNs) is poorly understood. Here we report genomic analyses of a patient with primary myelofibrosis (PMF) transformed to secondary acute myeloid leukemia (sAML). Whole genome sequencing (WGS) was performed on PMF and sAML diagnosis samples, with skin included as a germline surrogate. Deep sequencing validation was performed on the WGS samples and an additional sample obtained during sAML remission/relapsed PMF. Clustering analysis of 649 validated somatic single-nucleotide variants revealed four distinct clonal groups, each including putative driver mutations. The first group (including JAK2 and U2AF1), representing the founding clone, included mutations with high frequency at all three disease stages. The second clonal group (including MYB) was present only in PMF, suggesting the presence of a clone that was dispensable for transformation. The third group (including ASXL1) contained mutations with low frequency in PMF and high frequency in subsequent samples, indicating evolution of the dominant clone with disease progression. The fourth clonal group (including IDH1 and RUNX1) was acquired at sAML transformation and was predominantly absent at sAML remission/relapsed PMF. Taken together, these findings illustrate the complex clonal dynamics associated with disease evolution in MPNs and sAML.

The DNA double-strand break response is abnormal in myeloblasts from patients with therapy-related acute myeloid leukemia.

The complex chromosomal aberrations found in therapy-related acute myeloid leukemia (t-AML) suggest that the DNA double-strand break (DSB) response may be altered. In this study we examined the DNA DSB response of primary bone marrow cells from t-AML patients and performed next-generation sequencing of 37 canonical homologous recombination (HR) and non-homologous end-joining (NHEJ) DNA repair genes, and a subset of DNA damage response genes using tumor and paired normal DNA obtained from t-AML patients. Our results suggest that the majority of t-AML patients (11 of 15) have tumor-cell intrinsic, functional dysregulation of their DSB response. Distinct patterns of abnormal DNA damage response in myeloblasts correlated with acquired genetic alterations in TP53 and the presence of inferred chromothripsis. Furthermore, the presence of trisomy 8 in tumor cells was associated with persistently elevated levels of DSBs. Although tumor-acquired point mutations or small indels in canonical HR and NHEJ genes do not appear to be a dominant means by which t-AML leukemogenesis occurs, our functional studies suggest that an abnormal response to DNA damage is a common finding in t-AML.

Ectopic prostate: case report of a presacral mass presenting with obstructive symptoms.

We report the unusual case of a 78-year-old man who presented with obstructive bowel symptoms and a 2.5-cm presacral mass. The mass was excised and found on pathologic examination to be ectopic prostate tissue complete with a muscular stroma. Review of the literature revealed a number of case reports describing variably sized fragments of ectopic prostate tissue involving a variety of organs, including spleen, uterine cervix, bowel wall, pericolic fat, anal submucosa, seminal vesicle, testis, and urinary bladder. However, to our knowledge, this case is unique in that it presented as a relatively large, isolated presacral mass causing functional bowel impairment. The ectopic location can be related to the normal embryonic development of the prostate, rectum, and bladder.

Harmful freshwater algal blooms, with an emphasis on cyanobacteria.

Suspended algae, or phytoplankton, are the prime source of organic matter supporting food webs in freshwater ecosystems. Phytoplankton productivity is reliant on adequate nutrient supplies; however, increasing rates of nutrient supply, much of it manmade, fuels accelerating primary production or eutrophication. An obvious and problematic symptom of eutrophication is rapid growth and accumulations of phytoplankton, leading to discoloration of affected waters. These events are termed blooms. Blooms are a prime agent of water quality deterioration, including foul odors and tastes, deoxygenation of bottom waters (hypoxia and anoxia), toxicity, fish kills, and food web alterations. Toxins produced by blooms can adversely affect animal (including human) health in waters used for recreational and drinking purposes. Numerous freshwater genera within the diverse phyla comprising the phytoplankton are capable of forming blooms; however, the blue-green algae (or cyanobacteria) are the most notorious bloom formers. This is especially true for harmful toxic, surface-dwelling, scum-forming genera (e.g., Anabaena, Aphanizomenon, Nodularia, Microcystis) and some subsurface bloom-formers (Cylindrospermopsis, Oscillatoria) that are adept at exploiting nutrient-enriched conditions. They thrive in highly productive waters by being able to rapidly migrate between radiance-rich surface waters and nutrient-rich bottom waters. Furthermore, many harmful species are tolerant of extreme environmental conditions, including very high light levels, high temperatures, various degrees of desiccation, and periodic nutrient deprivation. Some of the most noxious cyanobacterial bloom genera (e.g., Anabaena, Aphanizomenon, Cylindrospermopsis, Nodularia) are capable of fixing atmospheric nitrogen (N2), enabling them to periodically dominate under nitrogen-limited conditions. Cyanobacteria produce a range of organic compounds, including those that are toxic to higher-ranked consumers, from zooplankton to further up the food chain. Both N2- and non-N2-fixing genera participate in mutualistic and symbiotic associations with microorganisms, higher plants, and animals. These associations appear to be of great benefit to their survival and periodic dominance. In this review, we address the ecological impacts and environmental controls of harmful blooms, with an emphasis on the ecology, physiology, and management of cyanobacterial bloom taxa. Combinations of physical, chemical, and biotic features of natural waters function in a synergistic fashion to determine the sensitivity of water bodies. In waters susceptible to blooms, human activities in water- and airsheds have been linked to the extent and magnitudes of blooms. Control and management of cyanobacterial and other phytoplankton blooms invariably includes nutrient input constraints, most often focused on nitrogen (N) and/or phosphorus (P). The types and amount of nutrient input constraints depend on hydrologic, climatic, geographic, and geologic factors, which interact with anthropogenic and natural nutrient input regimes. While single nutrient input constraints may be effective in some water bodies, dual N and P input reductions are usually required for effective long-term control and management of harmful blooms. In some systems where hydrologic manipulations (i.e., plentiful water supplies) are possible, reducing the water residence time by enhanced flushing and artificial mixing (in conjunction with nutrient input constraints) can be particularly effective alternatives. Implications of various management strategies, based on combined ecophysiological and environmental considerations, are discussed.

Comparative genomic sequence analysis of the human and mouse cystic fibrosis transmembrane conductance regulator genes.

The identification of the cystic fibrosis transmembrane conductance regulator gene (CFTR) in 1989 represents a landmark accomplishment in human genetics. Since that time, there have been numerous advances in elucidating the function of the encoded protein and the physiological basis of cystic fibrosis. However, numerous areas of cystic fibrosis biology require additional investigation, some of which would be facilitated by information about the long-range sequence context of the CFTR gene. For example, the latter might provide clues about the sequence elements responsible for the temporal and spatial regulation of CFTR expression. We thus sought to establish the sequence of the chromosomal segments encompassing the human CFTR and mouse Cftr genes, with the hope of identifying conserved regions of biologic interest by sequence comparison. Bacterial clone-based physical maps of the relevant human and mouse genomic regions were constructed, and minimally overlapping sets of clones were selected and sequenced, eventually yielding approximately 1.6 Mb and approximately 358 kb of contiguous human and mouse sequence, respectively. These efforts have produced the complete sequence of the approximately 189-kb and approximately 152-kb segments containing the human CFTR and mouse Cftr genes, respectively, as well as significant amounts of flanking DNA. Analyses of the resulting data provide insights about the organization of the CFTR/Cftr genes and potential sequence elements regulating their expression. Furthermore, the generated sequence reveals the precise architecture of genes residing near CFTR/Cftr, including one known gene (WNT2/Wnt2) and two previously unknown genes that immediately flank CFTR/Cftr.

Primary leiomyosarcoma of the vagina. A case report and literature review.

Primary vaginal leiomyosarcoma is a rare tumor. We report a unique case of a 27-year-old woman with stage I, high-grade primary leiomyosarcoma of the vagina treated with surgical resection and adjuvant radiation therapy. She returned within 6 months with an abdominal-pelvic recurrence and lung metastases. The patient died of disease 9 months after diagnosis. A comprehensive review of primary vaginal leiomyosarcoma was performed and factors affecting survival were analyzed. A Medline search of the English-language literature revealed 66 previously reported cases. Forty-eight of these had follow-up data. Survival probabilities were calculated using the Kaplan-Meier method, and the effects of age, stage, grade, tumor location, and treatment modality were analyzed. Stage III and IV data were combined. The overall 5-year survival rate was 43%. Patients more than 50 years of age had a 5-year survival rate of 26% compared with 51% for those less than 40 years. Five-year survival for stage I and II tumors was 55% and 44%, respectively. Patients with stage III/IV disease had 25% survival at 18 months. No patient treated primarily with chemotherapy or radiation therapy survived beyond 36 months. In contrast, patients treated primarily with surgery had a 5-year survival rate of 57%. Only stage remained an independent predictor of survival on Cox regression analysis. We continue to recommend surgical resection as primary treatment. Exenteration may be an option for select patients, but ultimately management should continue on a case-by-case basis.

A physical map of human chromosome 7: an integrated YAC contig map with average STS spacing of 79 kb.

The construction of highly integrated and annotated physical maps of human chromosomes represents a critical goal of the ongoing Human Genome Project. Our laboratory has focused on developing a physical map of human chromosome 7, a approximately 170-Mb segment of DNA that corresponds to an estimated 5% of the human genome. Using a yeast artificial chromosome (YAC)-based sequence-tagged site (STS)-content mapping strategy, 2150 chromosome 7-specific STSs have been established and mapped to a collection of YACs highly enriched for chromosome 7 DNA. The STSs correspond to sequences generated from a variety of DNA sources, with particular emphasis placed on YAC insert ends, genetic markers, and genes. The YACs include a set of relatively nonchimeric clones from a human-hamster hybrid cell line as well as clones isolated from total genomic libraries. For map integration, we have localized 260 STSs corresponding to Genethon genetic markers and 259 STSs corresponding to markers orders by radiation hybrid (RH) mapping on our YAC contigs. Analysis of the data with the program SEGMAP results in the assembly of 22 contigs that are "anchored" on the Genethon genetic map, the RH map, and/or the cytogenetic map. These 22 contigs are ordered relative to one another, are (in all but 3 cases) oriented relative to the centromere and telomeres, and contain > 98% of the mapped STSs. The largest anchored YAC contig, accounting for most of 7p, contains 634 STSs and 1260 YACs. An additional 14 contigs, accounting for approximately 1.5% of the mapped STSs, are assembled but remain unanchored on either the genetic or RH map. Therefore, these 14 "orphan" contigs are not ordered relative to other contigs. In our contig maps, adjacent STSs are connected by two or more YACs in > 95% of cases. With 2150 mapped STSs, our map provides an average STS spacing of approximately 79 kb. The physical map we report here exceeds the goal of 100-kb average STS spacing and should provide an excellent framework for systematic sequencing of the chromosome.

A collection of 1814 human chromosome 7-specific STSs.

An established goal of the ongoing Human Genome Project is the development and mapping of sequence-tagged sites (STSs) every 100 kb, on average, across all human chromosomes. En route to constructing such a physical map of human chromosome 7, we have generated 1814 chromosome 7-specific STSs. The corresponding PCR assays were designed by the use of DNA sequence determined in our laboratory (79%) or generated elsewhere (21%) and were demonstrated to be suitable for screening yeast artificial chromosome (YAC) libraries. This collection provides the requisite landmarks for constructing a physical map of chromosome 7 at < 100-kb average spacing of STSs.

A human chromosome 7 yeast artificial chromosome (YAC) resource: construction, characterization, and screening.

The paradigm of sequence-tagged site (STS)-content mapping involves the systematic assignment of STSs to individual cloned DNA segments. To date, yeast artificial chromosomes (YACs) represent the most commonly employed cloning system for constructing STS maps of large genomic intervals, such as whole human chromosomes. For developing a complete YAC-based STS-content map of human chromosome 7, we wished to utilize a limited set of YAC clones that were highly enriched for chromosome 7 DNA. Toward that end, we have assembled a human chromosome 7 YAC resource that consists of three major components: (1) a newly constructed library derived from a human-hamster hybrid cell line containing chromosome 7 as its only human DNA; (2) a chromosome 7-enriched sublibrary derived from the CEPH mega-YAC collection by Alu-polymerase chain reaction (Alu-PCR)-based hybridization; and (3) a set of YACs isolated from several total genomic libraries by screening for > 125 chromosome 7 STSs. In particular, the hybrid cell line-derived YACs, which comprise the majority of the clones in the resource, have a relatively low chimera frequency (10-20%) based on mapping isolated insert ends to panels of human-hamster hybrid cell lines and analyzing individual clones by fluorescence in situ hybridization. An efficient strategy for polymerase chain reaction (PCR)-based screening of this YAC resource, which totals 4190 clones, has been developed and utilized to identify corresponding YACs for > 600 STSs. The results of this initial screening effort indicate that the human chromosome 7 YAC resource provides an average of 6.9 positive clones per STS, a level of redundancy that should support the assembly of large YAC contigs and the construction of a high-resolution STS-content map of the chromosome.

Integration of physical, genetic and cytogenetic maps of human chromosome 7: isolation and analysis of yeast artificial chromosome clones for 117 mapped genetic markers.

An important goal for the human genome project is to assemble fully integrated physical, genetic and cytogenetic maps for each human chromosome. Towards that end, we have isolated yeast artificial chromosome (YAC) clones containing 117 of the 119 genetic markers that constitute a recently constructed, detailed genetic map of human chromosome 7. Analysis of these clones reveals numerous examples where adjacent genetic markers have been physically connected, either in individual YACs or in multi-YAC contigs. At present, the 117 genetic markers are contained in fewer than 80 YAC contigs, with most of these contigs uniquely ordered relative to one another based on the genetic map positions of the corresponding markers. These YACs and YAC contigs are estimated to contain approximately 60-85% of the DNA from human chromosome 7. YACs representing 36 genetic markers were mapped by fluorescence in situ hybridization (FISH) to metaphase chromosomes, allowing assignment of these genetic markers to cytogenetic bands along chromosome 7 and placement of the centromere within the genetic map. Together, these studies provide genetically and cytogenetically anchored YAC clones covering the majority of chromosome 7 that will be useful both for the positional cloning of genes and as a framework for assembling a complete YAC-based physical map of the chromosome.

Bond strength of repaired glass ionomer core materials.

Chelon Silver (C), Ketac-Silver Aplicap (K), Miracle Mix (M) silver-reinforced glass ionomers, and Valiant PH.D were evaluated as in vitro repair materials for cores. The core materials were placed in a 6 x 3 mm mold and stored at 37 degrees C at 100% RH for 24 hours. The cores were roughened with a coarse diamond. Five samples were conditioned (T) with Dentin Conditioner for 20 seconds, whereas another 5 samples were untreated (U). The cores were then repaired with C, K and M and subsequently tested after additional storage for 24 hours. Bond strength (MN/m2) was measured in tension using an inverted cone bond test at a crosshead speed of 0.05 cm/min. Mean bond strengths (S.D.) for the three repair materials when averaged over the three glass ionomer cores were: K, 5.0 (1.2); M, 3.4 (1.2) and C, 2.7 (1.5) for condition U; and M, 4.2 (1.1); K, 3.4 (1.2); and C, 3.1 (1.0) for condition T, where M, K and C were the repair materials. The Tukey intervals at the 95% level were 0.7 among materials and 0.5 between treatments. Bond strength of repaired amalgam was only successful with M, 0.9 MN/m2. Bond strengths to untreated silver-reinforced glass ionomer cores were higher with K used as a repair material.

Ultrastructural pulmonary changes in mice exposed to aerosolized Periodontal Pack Powder.

An electron microscopic study was undertaken to investigate pulmonary ultrastructural changes in mice following exposure to an aerosolized Periodontal Pack Powder (PPP), and to determine if the asbestos fiber, which contains 7% PPP, is respirable. One hundred and sixty-five animals were used in this study. Seventy-two of the animals were exposed to the powder twice weekly for a minimum of two weeks and a maximum of four weeks. Fifty animals were exposed to the PPP (without asbestos), with the same exposure sequence used. Forty-three animals served as nonexposed controls. Randomly selected groups of exposed and control mice were killed at designated intervals between two and 12 weeks. Microscopic examination of the lungs revealed early interstitial pneumonia. These changes were seen in both treatment groups. Late changes included a generalized resolution of the pneumonia (seen in both treatment groups) with residual diffuse interstitial fibrosis, persistence of asbestos fibers within the tissue, and early asbestos body formation (seen only in animals exposed to asbestos-containing PPP). This study documents that the asbestos in Periodontal Pack Powder is both respirable and potentially pathogenic. Dentists who have used this product could conceivably be a population at risk for pulmonary fibrosis and/or obstructive airways disease.

Effects of the blue-green alga Microcystis aeruginosa on zooplankton competitive relations.

Field distribution patterns and laboratory feeding experiments have suggested that blooms of colonial blue-green algae strongly inhibit relatively large-bodied daphnid cladocerans. We conducted laboratory experiments to test the hypothesis that blooms of the colonial blue-green alga Microcystis aeruginosa would shift competitive dominance away from large-bodied daphnid cladocerans toward smaller-bodied cladocerans, copepods, and rotifers. In laboratory competition experiments, increasing the proportion of M. aeruginosa in the algal food supply resulted in a shift from dominance by the relatively largebodied cladoceran Daphnia ambigua to dominace by the copepod Diaptomus reighardi. The small-bodied cladoceran Bosmina longirostris was always numerically heavily dominant over D. ambigua, but its estimated population biomasses were only slightly higher than those of D. ambigua. Daphnia ambigua consistently outcompeted the rotifer Brachionus calyciflorus. Our results demonstrate that blooms of M. aeruginosa can alter zooplankton competitive relations in laboratory experiments, favoring small-bodied cladocerans and copepods at the expense of large-bodied cladocerans. However, contrary to predictions, blooms of M. aeruginosa did not improve the competitive ability of rotifers.

Effects of chaetognath predation and nutrient enrichment on enclosed estuarine copepod communities.

The effects of predation by the chaetognath Sagitta hispida and nutrient enrichment on estuarine copepod community structure were studied by experimental manipulations in large enclosures. Chaetognath abundance and nutrient addition rates were manipulated in a factorial design. Predation by chaetognaths resulted in a significant decline in the relative abundance of Acartia tonsa and an increase in the relative abundance of Oithona colcarva. However, these effects were evident only at chaetognath densities far higher than observed in natural populations. Nutrient enrichment resulted in a decline in relative abundance of Paracalanus crassirostris, and, in the absence of chaetognath predation, an apparent increase in the relative abundance of A. tonsa. The effects of chaetognath predation were independent of enrichment level, apparently because of the absence of effects of enrichment on total copepod and chaetognath densities.