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Bone and muscle are coupled through developmental, mechanical, paracrine, and autocrine signals. Genetic variants at the CPED1-WNT16 locus are dually associated with bone- and muscle-related traits. While Wnt16 is necessary for bone mass and strength, this fails to explain pleiotropy at this locus. Here, we show wnt16 is required for spine and muscle morphogenesis in zebrafish. In embryos, wnt16 is expressed in dermomyotome and developing notochord, and contributes to larval myotome morphology and notochord elongation. Later, wnt16 is expressed at the ventral midline of the notochord sheath, and contributes to spine mineralization and osteoblast recruitment. Morphological changes in wnt16 mutant larvae are mirrored in adults, indicating that wnt16 impacts bone and muscle morphology throughout the lifespan. Finally, we show that wnt16 is a gene of major effect on lean mass at the CPED1-WNT16 locus. Our findings indicate that Wnt16 is secreted in structures adjacent to developing bone (notochord) and muscle (dermomyotome) where it affects the morphogenesis of each tissue, thereby rendering wnt16 expression into dual effects on bone and muscle morphology. This work expands our understanding of wnt16 in musculoskeletal development and supports the potential for variants to act through WNT16 to influence bone and muscle via parallel morphogenetic processes.

Bone and muscle interact through developmental, mechanical, paracrine, and autocrine signals. Genetic variants at the CPED1-WNT16 locus are dually associated with bone- and muscle-related traits. While Wnt16 is necessary for bone mass and strength, this fails to explain pleiotropy at this locus. Here, we show wnt16 is required for spine and muscle morphogenesis in zebrafish. In embryos, wnt16 is expressed in dermomyotome and developing notochord, and contributes to larval myotome morphology and notochord elongation. Later, wnt16 is expressed at the ventral midline of the notochord sheath, and contributes to spine mineralization and osteoblast recruitment. Morphological changes in wnt16 mutant larvae are mirrored in adults, indicating that wnt16 impacts bone and muscle morphology throughout the lifespan. Finally, we show that wnt16 is a gene of major effect on lean mass at the CPED1-WNT16 locus. Our findings indicate that Wnt16 is secreted in structures adjacent to developing bone (notochord) and muscle (dermomyotome) where it affects the morphogenesis of each tissue, thereby rendering wnt16 expression into dual effects on bone and muscle morphology. This work expands our understanding of wnt16 in musculoskeletal development and supports the potential for variants to act through WNT16 to influence bone and muscle via parallel morphogenetic processes.AUTHOR SUMMARYIn humans, genetic variants (DNA sequences that vary amongst individuals) have been identified that appear to influence bone and skeletal muscle mass. However, how single genes and genetic variants exert dual influence on both tissues is not well understood. In this study, we found a gene called wnt16 is necessary for specifying the size and shape of muscle and bone during development in zebrafish. Moreover, we disentangled how wnt16 affects both tissues: distinct cellular populations adjacent to muscle and bone secrete Wnt16, where it acts as a signal this guides the size and shape of each tissue. This is important because in humans, genetic variants near the WNT16 gene have effects on both bone- and muscle-related traits. This study expands our understanding of the role of WNT16 in bone and muscle development, and helps to explain how genetic variants near WNT16 affect traits for both tissues. Moreover, WNT16 is actively being explored as a target for osteoporosis therapies; our study could have implications with regard to the potential to target WNT16 to treat bone and muscle simultaneously.

Background Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder and is one of the most common muscular dystrophies. There are currently few effective therapies to treat the disease, although many small-molecule approaches are being pursued. Certain histone deacetylase inhibitors (HDACi) have been shown to ameliorate DMD phenotypes in mouse and zebrafish animal models. The HDACi givinostat has shown promise for DMD in clinical trials. However, beyond a small group of HDACi, other classes of epigenetic small molecules have not been broadly and systematically studied for their benefits for DMD. Methods We used an established animal model for DMD, the zebrafish dmd mutant strain sapje. A commercially available library of epigenetic small molecules was used to treat embryonic-larval stages of dmd mutant zebrafish. We used a quantitative muscle birefringence assay in order to assess and compare the effects of small-molecule treatments on dmd mutant zebrafish skeletal muscle structure. Results We performed a novel chemical-combination screen of a library of epigenetic compounds using the zebrafish dmd model. We identified candidate pools of epigenetic compounds that improve skeletal muscle structure in dmd mutant zebrafish. We then identified a specific combination of two HDACi compounds, oxamflatin and salermide, that ameliorated dmd mutant zebrafish skeletal muscle degeneration. We validated the effects of oxamflatin and salermide on dmd mutant zebrafish in an independent laboratory. Furthermore, we showed that the combination of oxamflatin and salermide caused increased levels of histone H4 acetylation in zebrafish larvae. Conclusions Our results provide novel, effective methods for performing a combination of small-molecule screen in zebrafish. Our results also add to the growing evidence that epigenetic small molecules may be promising candidates for treating DMD.

SummaryVertebrate skeletal muscles are composed of both slow-twitch and fast-twitch fiber types. How the differentiation of distinct fiber types is activated during embryogenesis is not well characterized. Skeletal muscle differentiation is initiated by the activity of the myogenic basic helix-loop-helix (bHLH) transcription factors Myf5, Myod1, Myf6, and Myog. Myod1 functions as a muscle master regulatory factor and directly activates muscle differentiation genes, including those specific to both slow and fast muscle fibers. Our previous studies showed that Pbx TALE-class homeodomain proteins bind with Myod1 on the promoter of the zebrafish fast muscle genemylpfaand are required for proper activation ofmylpfaexpression and the fast-twitch muscle-specific differentiation program in zebrafish embryos. Pbx proteins have also been shown to bind regulatory regions of muscle differentiation genes in mammalian muscle cells in culture. Here, we use new zebrafish mutant strains to confirm the essential roles of zebrafish Pbx factors in embryonic fast muscle differentiation. Furthermore, we examine the requirements forPbxgenes in mouse embryonic skeletal muscle differentiation, an area that has not been investigated in the mammalian embryo. RemovingPbx1function from skeletal muscle inMyf5Cre/+;Pbx1fl/flmouse embryos has minor effects on embryonic muscle development. However, concomitantly deletingPbx2function inMyf5Cre/+;Pbx1fl/fl;Pbx2-/-mouse embryos causes delayed activation and reduced expression of fast muscle differentiation genes. In the mouse,Pbx1/Pbx2-dependent fast muscle genes closely match those that have been previously shown to be dependent on murineSix1andSix4. This work establishes evolutionarily conserved requirements for Pbx factors in embryonic fast muscle differentiation. Our studies are revealing how Pbx homeodomain proteins help direct specific cellular differentiation pathways.

SummaryDuchenne muscular dystrophy (DMD) is a severe neuromuscular disorder and is one of the most common muscular dystrophies. There are currently few effective therapies to treat the disease, although many small-molecule approaches are being pursued. Specific histone deacetylase inhibitors (HDACi) can ameliorate DMD phenotypes in mouse and zebrafish animal models and have also shown promise for DMD in clinical trials. However, beyond these HDACi, other classes of epigenetic small molecules have not been broadly and systematically studied for their benefits for DMD. Here, we performed a novel chemical screen of a library of epigenetic compounds using the zebrafishdmdmodel. We identified candidate pools of epigenetic compounds that improve skeletal muscle structure indmdzebrafish. We then identified a specific combination of two drugs, oxamflatin and salermide, that significantly rescueddmdzebrafish skeletal muscle degeneration. Furthermore, we validated the effects of oxamflatin and salermide in an independent laboratory. Our results provide novel, effective methods for performing a combination small-molecule screen in zebrafish. Our results also add to the growing evidence that epigenetic small molecules may be promising candidates for treating DMD.

The mesodermal germ layer is patterned into mediolateral subtypes by signaling factors including BMP and FGF. How these pathways are integrated to induce specific mediolateral cell fates is not well understood. We used mesoderm derived from post-gastrulation neuromesodermal progenitors (NMPs), which undergo a binary mediolateral patterning decision, as a simplified model to understand how FGF acts together with BMP to impart mediolateral fate. Using zebrafish and mouse NMPs, we identify an evolutionarily conserved mechanism of BMP and FGF-mediated mediolateral mesodermal patterning that occurs through modulation of basic helix-loop-helix (bHLH) transcription factor activity. BMP imparts lateral fate through induction of Id helix loop helix (HLH) proteins, which antagonize bHLH transcription factors, induced by FGF signaling, that specify medial fate. We extend our analysis of zebrafish development to show that bHLH activity is responsible for the mediolateral patterning of the entire mesodermal germ layer.

Whole-genome and exome sequencing efforts are increasingly identifying candidate genetic variants associated with human disease. However, predicting and testing the pathogenicity of a genetic variant remains challenging. Genome editing allows for the rigorous functional testing of human genetic variants in animal models. Congenital heart defects (CHDs) are a prominent example of a human disorder with complex genetics. An inherited sequence variant in the human PBX3 gene (PBX3 p.A136V) has previously been shown to be enriched in a CHD patient cohort, indicating that the PBX3 p.A136V variant could be a modifier allele for CHDs. Pbx genes encode three-amino-acid loop extension (TALE)-class homeodomain-containing DNA-binding proteins with diverse roles in development and disease, and are required for heart development in mouse and zebrafish. Here, we used CRISPR-Cas9 genome editing to directly test whether this Pbx gene variant acts as a genetic modifier in zebrafish heart development. We used a single-stranded oligodeoxynucleotide to precisely introduce the human PBX3 p.A136V variant in the homologous zebrafish pbx4 gene (pbx4 p.A131V). We observed that zebrafish that are homozygous for pbx4 p.A131V are viable as adults. However, the pbx4 p.A131V variant enhances the embryonic cardiac morphogenesis phenotype caused by loss of the known cardiac specification factor, Hand2. Our study is the first example of using precision genome editing in zebrafish to demonstrate a function for a human disease-associated single nucleotide variant of unknown significance. Our work underscores the importance of testing the roles of inherited variants, not just de novo variants, as genetic modifiers of CHDs. Our study provides a novel approach toward advancing our understanding of the complex genetics of CHDs., Summary: This study uses genome editing in zebrafish to demonstrate that a human DNA sequence variant of unknown significance might contribute to the complex genetics of congenital heart defects.

Lipid nanoparticles (LNPs) are an attractive platform for the delivery of therapeutic RNA molecules because LNPs are versatile, have been validated in clinical trials, and are well tolerated. Here, we test whether LNPs can be used to deliver a reporter green fluorescent protein (gfp) mRNA to different tissues in zebrafish embryos. We show that LNP-packaged gfp mRNA can be delivered, through injection, and taken up by cells in multiple tissues in zebrafish embryos without any apparent detrimental effects on embryonic health or survival. Zebrafish embryos injected with LNP-packaged gfp mRNA show subsequent GFP expression in neural, vascular, cardiac, and skeletal muscle tissue, depending on injection site. In contrast, comparable naked (nonpackaged) gfp mRNA injections lead to little or no GFP expression. This study shows that LNPs can be used as an mRNA delivery platform in zebrafish and thus provides a basis for testing the therapeutic functions of LNP-packaged candidate mRNAs in the increasingly diverse array of zebrafish disease models.

Summary The basic helix–loop–helix factor Myod initiates skeletal muscle differentiation by directly and sequentially activating sets of muscle differentiation genes, including those encoding muscle contractile proteins. We hypothesize that Pbx homeodomain proteins direct Myod to a subset of its transcriptional targets, in particular fast-twitch muscle differentiation genes, thereby regulating the competence of muscle precursor cells to differentiate. We have previously shown that Pbx proteins bind with Myod on the promoter of the zebrafish fast muscle gene mylpfa and that Pbx proteins are required for Myod to activate mylpfa expression and the fast-twitch muscle-specific differentiation program in zebrafish embryos. Here we have investigated the interactions of Pbx with another muscle fiber-type regulator, Prdm1a, a SET-domain DNA-binding factor that directly represses mylpfa expression and fast muscle differentiation. The prdm1a mutant phenotype, early and increased fast muscle differentiation, is the opposite of the Pbx-null phenotype, delayed and reduced fast muscle differentiation. To determine whether Pbx and Prdm1a have opposing activities on a common set of genes, we used RNA-seq analysis to globally assess gene expression in zebrafish embryos with single- and double-losses-of-function for Pbx and Prdm1a. We find that the levels of expression of certain fast muscle genes are increased or approximately wild type in pbx2/4-MO;prdm1a−/− embryos, suggesting that Pbx activity normally counters the repressive action of Prdm1a for a subset of the fast muscle program. However, other fast muscle genes require Pbx but are not regulated by Prdm1a. Thus, our findings reveal that subsets of the fast muscle program are differentially regulated by Pbx and Prdm1a. Our findings provide an example of how Pbx homeodomain proteins act in a balance with other transcription factors to regulate subsets of a cellular differentiation program.

Few effective options are available for the treatment of unresectable hepatocellular carcinoma (HCC). Several phase I trials suggest promising activity of a combination of gemcitabine and docetaxel.Patients with unresectable or metastatic HCC were treated with docetaxel 40 mg/m (later reduced to 30 mg/m) and gemcitabine 800 mg/m on days 1 and 8 every 3 weeks. Twenty-five patients were enrolled in 26 months. Median age was 64 (range, 27-078), 17 were male, 14 had liver-only disease, and 11 had extrahepatic disease.Of 25 patients evaluable for the primary end point (response), 2 (8%) have a confirmed partial response. The median time to progression is 2.76 months (95% confidence interval, 1.84-6.64 mo). Median survival was 12.8 months (95% confidence interval, 5.26-28.00). Two patients died on study owing to adverse events (1 hepatic and 1 renal failure), neither of which were attributed to the study medications. Twenty patients (81%) have experienced grade 3+ adverse events, including 11 with grade 4+ adverse events, primarily neutropenia, thrombocytopenia, diarrhea, and fatigue.Although this combination seems to have potential benefit, as measured by overall survival, its toxicity and the recent introduction of sorafenib has further limited the use of chemotherapy. Approaches other than chemotherapy are likely to be of the greatest potential benefit.

Proper control of the temporal onset of cellular differentiation is critical for regulating cell lineage decisions and morphogenesis during development. Pbx homeodomain transcription factors have emerged as important regulators of cellular differentiation. We previously showed, by using antisense morpholino knockdown, that Pbx factors are needed for the timely activation of myocardial differentiation in zebrafish. In order to gain further insight into the roles of Pbx factors in heart development, we show here that zebrafish pbx4 mutant embryos exhibit delayed onset of myocardial differentiation, such as delayed activation of tnnt2a expression in early cardiomyocytes in the anterior lateral plate mesoderm. We also observe delayed myocardial morphogenesis and dysmorphic patterning of the ventricle and atrium, consistent with our previous Pbx knock-down studies. In addition, we find that pbx4 mutant larvae have aberrant outflow tracts and defective expression of the proepicardial marker tbx18. Finally, we present evidence for Pbx expression in cardiomyocyte precursors as well as heterogeneous Pbx expression among the pan-cytokeratin-expressing proepicardial cells near the developing ventricle. In summary, our data show that Pbx4 is required for the proper temporal activation of myocardial differentiation and establish a basis for studying additional roles of Pbx factors in heart development.

SummaryMyoD and NeuroD2, master regulators of myogenesis and neurogenesis, bind to a “shared” E-box sequence (CAGCTG) and a “private” sequence (CAGGTG or CAGATG, respectively). To determine whether private-site recognition is sufficient to confer lineage specification, we generated a MyoD mutant with the DNA-binding specificity of NeuroD2. This chimeric mutant gained binding to NeuroD2 private sites but maintained binding to a subset of MyoD-specific sites, activating part of both the muscle and neuronal programs. Sequence analysis revealed an enrichment for PBX/MEIS motifs at the subset of MyoD-specific sites bound by the chimera, and point mutations that prevent MyoD interaction with PBX/MEIS converted the chimera to a pure neurogenic factor. Therefore, redirecting MyoD binding from MyoD private sites to NeuroD2 private sites, despite preserved binding to the MyoD/NeuroD2 shared sites, is sufficient to change MyoD from a master regulator of myogenesis to a master regulator of neurogenesis.

We conducted this study to provide one of the initial assessments of treatment outcomes for breast cancer patients evaluated with sentinel node mapping. All patients diagnosed with breast carcinoma, evaluated with sentinel node mapping, and followed for 5 years were divided into three groups depending on sentinel node(s) status. Group I (node negative) included 91 patients, 77 with invasive cancer, and 7 lost to follow-up. Of the remaining 70 patients, 3 (4.3%) suffered a distant recurrence and died, 1 developed an in-breast recurrence, and 9 (12.9%) developed a contralateral cancer during the study. Group II (IHC positive) included 28 patients. One (3.6%) developed a distant recurrence and died of breast cancer, and one developed a contralateral cancer during follow. Group III (H&E positive) included 36 patients with 1 lost to follow-up. Five patients (14.3%) died of breast cancer and two (5.7%) developed contralateral carcinomas during follow-up. The most striking observation was a lower than expected rate of distant recurrences in these patients followed for 5 years after a diagnosis of breast cancer and staging with sentinel node mapping. The ability to identify subtle nodal metastasis and design appropriate systemic therapeutic strategies may explain this finding.

Purpose This Phase III study was performed to determine whether twice-daily (b.i.d.) radiotherapy (RT) resulted in better survival than once-daily (q.d.) RT for patients with limited-stage small-cell lung cancer (LD-SCLC). Methods and materials A total of 310 patients with LD-SCLC initially received three cycles of etoposide and cisplatin. Subsequently, the 261 patients without significant progression were randomized to two cycles of etoposide and cisplatin plus either q.d. RT (50.4 Gy in 28 fractions) or split-course b.i.d. RT (24 Gy in 16 fractions, a 2.5-week break, and 24 Gy in 16 fractions) to the chest. Patients then received a sixth cycle of etoposide and cisplatin followed by prophylactic cranial RT. Results Follow-up ranged from 4.6 to 11.9 years (median, 7.4 years). The median survival and 5-year survival rate from randomization was 20.6 months and 21% for patients who received q.d. RT compared with 20.6 months and 22% for those who received b.i.d. RT (p = 0.68), respectively. No statistically significant differences were found in the rates of progression (p = 0.68), intrathoracic failure (p = 0.45), in-field failure (p = 0.62), or distant failure (p = 0.82) between the two treatment arms. No statistically significant difference was found in the overall rate of Grade 3 or worse (p = 0.83) or Grade 4 or worse toxicity (p = 0.95). Grade 3 or worse esophagitis (p = 0.05) was more common in the b.i.d. arm. Grade 5 toxicity occurred in 4 (3%) of 130 patients who received b.i.d. RT compared with 0 (0%) of 131 who received q.d. RT (p = 0.04). Conclusion Although this study did not demonstrate an advantage to split-course b.i.d. RT, the long-term survival was favorable, likely reflecting the positive influences of concurrent combined modality therapy and prophylactic cranial RT.

Anterior to posterior growth of the vertebrate body is fueled by a posteriorly located population of bipotential neuro-mesodermal progenitor cells. These progenitor cells have a limited rate of proliferation, and their maintenance is critical for completion of the anterior-posterior axis. How these cells leave the progenitor state and commit to differentiation is largely unknown, in part because widespread modulation of factors essential for this process causes organism-wide effects. Using a novel assay, we show that Tbx16 (Spadetail) is capable of advancing mesodermal differentiation cell-autonomously. We find that Tbx16 locks cells into the mesodermal state by not only activating downstream mesodermal genes, but also by repressing bipotential progenitor genes, in part through a direct repression of sox2. We demonstrate that tbx16 is activated as cells move from an intermediate Wnt environment to a high Wnt environment, and show that Wnt signaling activates the tbx16 promoter. Importantly, high-level Wnt signaling is able to accelerate mesodermal differentiation cell-autonomously, just as we observe with Tbx16. Finally, because our assay for mesodermal commitment is quantitative, we show that the acceleration of mesodermal differentiation is surprisingly incomplete, implicating a potential separation of cell movement and differentiation during this process. Together our data suggest a model in which high levels of Wnt signaling induce a transition to mesoderm by directly activating tbx16, which in turn acts to irreversibly flip a bistable switch, leading to maintenance of the mesodermal fate and repression of the bipotential progenitor state, even as cells leave the initial high Wnt environment.

An image-guided core-needle breast biopsy (IGCNBB) diagnosis of ductal carcinoma in situ (DCIS) is often upgraded to invasive carcinoma (IC) after complete excision. When IC is identified after excision patients must be returned to the operating room for evaluation of their axillary nodes. We performed this study to identify histologic or mammographic features that would predict the presence of invasion when DCIS is documented by IGCNBB. Patients with an IGCNBB diagnosis of DCIS were identified from a prospective database. Imaging abnormalities were classified as either calcification only or mass with or without calcifications. IGCNBB specimens were reviewed to document nuclear grade and the presence of comedo-type necrosis, periductal fibrosis, and periductal inflammation. Patients were divided into two groups, DCIS and IC, on the basis of the final diagnosis after complete excision. From July 1993 through May 2000, 148 of 2995 (4.9%) IGCNBBs demonstrated DCIS; eight were excluded after pathologic review. Of the remaining 140 patients 36 (26%) demonstrated IC after complete excision. The presence of a mass on breast imaging was the only significant predictor of IC ( P = 0.04). On the basis of the results of this study we now perform sentinel lymph node mapping and biopsy at the initial surgical procedure for patients with an IGCNBB diagnosis of DCIS and an associated mass on breast imaging.

To understand how the Wnt coreceptor LRP-5 is involved in transducing the canonical Wnt signals, we identified Axin as a protein that interacts with the intracellular domain of LRP-5. LRP-5, when expressed in fibroblast cells, showed no effect on the canonical Wnt signaling pathway by itself, but acted synergistically with Wnt. In contrast, LRP-5 mutants lacking the extracellular domain functioned as constitutively active forms that bind Axin and that induce LEF-1 activation by destabilizing Axin and stabilizing beta-catenin. Addition of Wnt caused the translocation of Axin to the membrane and enhanced the interaction between Axin and LRP-5. In addition, the LRP-5 sequences involved in interactions with Axin are required for LEF-1 activation. Thus, we conclude that the binding of Axin to LRP-5 is an important part of the Wnt signal transduction pathway.

Image-guided core needle breast biopsy (IGCNBB) is an incisional biopsy technique that has been associated with tumor cell displacement. Theoretically tumor cell displacement may affect local recurrence rates in patients treated with breast-conserving therapy (BCT). We performed a study to determine if the biopsy method impacted local control rates following BCT. Patients with nonpalpable breast cancer (invasive and intraductal) diagnosed at our institution and treated with BCT between July 1993 and July 1996 were selected to provide a follow-up period in which the majority of local recurrences should be detected. Patients were divided into two groups based on their method of diagnosis. Group I patients were diagnosed by IGCNBB and group II patients were diagnosed by wire localized excisional breast biopsy (WLEBB). Factors potentially affecting local recurrence rates were retrospectively reviewed. Two hundred eleven patients were treated with BCT, 132 were diagnosed by IGCNBB and 79 by WLEBB. The two patient groups were similar when compared for prognostic factors and treatment. All patients' BCT included histologically negative margins. There were 4 (3.0%) local recurrences in Group I at a median follow-up of 44.4 months and 2 (2.5%) local recurrences in group II at a median follow-up of 50.1 months. This difference was not significant. Breast cancer patients diagnosed by IGCNBB can be treated by BCT with acceptable local control rates. Additional surveillance of our institutional experience and others' is mandatory to validate IGCNBB as the preferred biopsy method for nonpalpable mammographic abnormalities.

Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is b -catenin, a downstream ef- fector of the Wnt signaling pathway that is required for dorsal-ventral axis specification in the Xenopus em- bryo. GSK-3 activity is regulated through the opposing activities of multiple proteins. Axin, GSK-3, and b -cate- nin form a complex that promotes the GSK-3-medi- ated phosphorylation and subsequent degradation of b -catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates b -catenin in mammalian cells, but its role in Xenopus is less clear. In contrast, GBP, which is required for axis formation in Xenopus , binds and inhibits GSK-3. We show here that GSK-3 binding protein (GBP) inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, we present evidence that a dominant-negative GSK-3 mu- tant, which causes the same effects as GBP, keeps en- dogenous GSK-3 from binding to Axin. We show that GBP also functions by preventing the GSK-3-mediated phosphorylation of a protein substrate without elimi- nating its catalytic activity. Finally, we show that the previously demonstrated axis-inducing property of overexpressed APC is attributable to its ability to stabi- lize cytoplasmic b -catenin levels, demonstrating that APC is impinging upon the canonical Wnt pathway in this model system. These results contribute to our growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in b -catenin levels and establishment of the dorsal axis.

Image-guided core-needle breast biopsy (IGCNBB) is widely used to evaluate patients with abnormal mammograms; however, information is limited regarding the reliability of a benign diagnosis. The goal of this study was to demonstrate that a benign diagnosis obtained by IGCNBB is accurate and amenable to mammographic surveillance. Records of all patients evaluated by IGCNBB from July 1993 through July 1996 were reviewed. Biopsies were classified as malignant, atypical, or benign. All benign cases were followed by surveillance mammography beginning 6 months after IGCNBB. Of the 1110 patients evaluated by IGCNBB during the study period, 855 revealed benign pathology. A total of 728 of the 855 patients (85%) complied with the recommendation for surveillance mammography. A total of 196 IGCNBBs were classified as malignant; 59 cases were classified as atypical. The atypical cases were excluded from the statistical analysis. Only two patients have demonstrated carcinoma after a benign IGCNBB during the 2-year minimum follow-up period. The sensitivity and specificity of a benign result were 100.0 and 98.9 per cent, respectively. A benign diagnosis obtained by IGCNBB is accurate and therefore amenable to mammographic surveillance. The results of this study support IGCNBB as the preferred method of evaluating women with abnormal mammograms.

Hepatitis B after liver transplantation is often fatal, and no proven medical therapy exists for this condition. We chose to study the potential efficacy of lamivudine therapy for patients with chronic hepatitis B after liver transplantation. Fifty-two patients with chronic hepatitis B after liver transplantation were treated in an open label, multicenter study. Each had detectable hepatitis B virus (HBV) DNA in serum and 45 (87%) had detectable serum hepatitis B e antigen before treatment. Patients were treated for 52 weeks with lamivudine (100 mg daily). The primary endpoint was undetectability of HBV DNA; secondary endpoints included normalization of serum alanine transaminase (ALT) levels, disappearance of hepatitis B e antigen, and improvement in liver histology. After treatment, 60% of patients had undetectable HBV DNA by solution hybridization assay, 14 (31%) of the initially positive patients lost hepatitis B e antigen; hepatitis B surface antigen was undetectable in 3 (6%); and serum ALT levels normalized in 71%. Blinded histological assessments showed improvement in the histological activity index (P =.007 for periportal necrosis,.001 for lobular necrosis, and.013 for portal inflammation). YMDD variants of HBV, potentially associated with drug resistance, were detected in 14 (27%) of the patients. Repeat liver biopsies in 7 patients with the mutated virus were unchanged in 2, improved in 2, and worse in 3. We conclude that lamivudine is a potentially effective therapy for hepatitis B after liver transplantation.

Continuous mucosal involvement from the rectum proximally is one of the hallmarks of ulcerative colitis. However, recent pathologic series report appendiceal ulcerative colitis in the presence of a histologically normal cecum, representing a "skip" lesion. The clinical significance of this finding has not been established. Eighty patients, 54 males and 26 females, average age 37.9 years (range 14 to 82 years) who underwent proctocolectomy for ulcerative colitis from January 1990 to September 1995 were examined to determine the rate of discontinuous appendiceal involvement. Excluded were 12 patients with prior appendectomy and 11 with fibrotic obliteration of the appendiceal lumen. Of the remaining 57 patients, seven (12.3%) had clear appendiceal involvement in the presence of a histologically normal cecum. These seven patients clinically were indistinguishable from the 50 patients without skip involvement of the appendix in terms of age at surgery, pretreatment medications, type of surgery, interval from diagnosis to definitive procedure, complications, functional results, and clinical course. Discontinuous appendiceal involvement was found in 12.3% of patients undergoing proctocolectomy for ulcerative colitis, and clinically these patients behave as those without this feature.

The prognostic significance of neuroendocrine differentiation in colorectal carcinoma is uncertain. We analyzed 289 moderately differentiated (grades II and Ill) colorectal carcinomas for neuroendocrine differentiation by immunohistochemistry and in situ hybridization. The tumors were divided into three groups based on the presence of and the numbers of neuroendocrine cells, with group I having no neuroendocrine cells, group II having1 positive cell/mm(2), and group Ill with1 positive cell/mm(2). In situ hybridization with probes for chromogranin A and B detected almost twice as many neuroendocrine cells as did immunostaining with an antibody for chromogranin A. There was no prognostic difference associated with the presence or absence of neuroendocrine differentiation in this group of moderately differentiated carcinomas. These results indicate that the presence of neuroendocrine cells detected by expression of chromogranin protein or mRNA does not influence prognosis in moderately differentiated colorectal carcinomas.

The vertebrate body forms from a multipotent stem cell-like progenitor population that progressively contributes newly differentiated cells to the most posterior end of the embryo. How the progenitor population balances proliferation and other cellular functions is unknown due to the difficulty of analyzing cell division in vivo. Here, we show that proliferation is compartmentalized at the posterior end of the embryo during early zebrafish development by the regulated expression of cdc25a, a key controller of mitotic entry. Through the use of a transgenic line that misexpresses cdc25a, we show that this compartmentalization is critical for the formation of the posterior body. Upon misexpression of cdc25a, several essential T-box transcription factors are abnormally expressed, including Spadetail/Tbx16, which specifically prevents the normal onset of myoD transcription, leading to aberrant muscle formation. Our results demonstrate that compartmentalization of proliferation during early embryogenesis is critical for both extension of the vertebrate body and differentiation of the multipotent posterior progenitor cells to the muscle cell fate.

Stereotactic core needle breast biopsy (SCNBB) is a minimally invasive technique used to sample nonpalpable mammographic abnormalities. The optimal management of atypical ductal hyperplasia (ADH) diagnosed by SCNBB is unknown. We hypothesized that ADH diagnosed by SCNBB should be evaluated by excisional breast biopsy (EBB) because of the risk of identifying carcinoma in association with ADH that would be missed if a diagnostic sampling technique alone was utilized.To test this hypothesis, a prospective diagnostic protocol was created which called for SCNBB instead of EBB for patients with mammographic abnormalities considered suspicious for malignancy. If ADH was noted on histologic evaluation of the cores, patients were advised to undergo an EBB.A review of the initial 900 patients evaluated by SCNBB yielded 39 patients (4.3%) with ADH detected by SCNBB. Thirty-six of these 39 patients agreed to proceed with EBB: 19 patients demonstrated benign findings including atypical ductal hyperplasia, 13 patients demonstrated noninvasive ductal carcinoma, and 4 patients had evidence of invasive carcinoma.A 47% rate of detecting noninvasive or invasive breast carcinoma supports the hypothesis that ADH detected by a sampling technique, such as SCNBB, should be managed by EBB.

Background Two arms with FOLFIRI, with or without cetuximab, were initially included in the randomized phase III intergroup clinical trial NCCTG (North Central Cancer Treatment Group) N0147. When other contemporary trials demonstrated no benefit to using irinotecan as adjuvant therapy, the FOLFIRI-containing arms were discontinued. We report the clinical outcomes for patients randomized to FOLFIRI with or without cetuximab. Patients and Methods After resection, patients were randomized to 12 biweekly cycles of FOLFIRI, with or without cetuximab. KRAS (Kirsten rat sarcoma viral oncogene homolog) mutation status was retrospectively determined in a central lab. The primary end point was disease-free survival (DFS). Secondary end points included overall survival (OS) and toxicity. Results One hundred and six patients received FOLFIRI and 40 received FOLFIRI plus cetuximab. Median follow-up was 5.95 years (range, 0.1-7.0 years). The addition of cetuximab showed a trend toward improved DFS (hazard ratio [HR], 0.53; 95% CI, 0.26-1.1; P = .09) and OS (HR, 0.45; 95% CI, 0.17-1.16; P = .10) in the overall group, regardless of KRAS status, and in patients with wild type KRAS . Grade ≥ 3 nonhematologic adverse effects were significantly increased in the cetuximab versus FOLFIRI-alone arm (68% vs. 46%; P = .02). Adjuvant FOLFIRI resulted in a 3-year DFS less than that expected for FOLFOX. Conclusion In this small randomized subset of patients with resected stage III colon cancer, the addition of cetuximab to FOLFIRI was associated with a nonsignificant trend toward improved DFS and OS. Nevertheless, considering the limitations of this analysis, FOLFOX without the addition of a biologic agent remains the standard of care for adjuvant therapy in resected stage III colon cancer.

OBJECTIVE: Our purpose was to evaluate the importance of deoxyribonucleic acid content to long-term survival from advanced epithelial ovarian carcinoma. STUDY DESIGN: Clinical and pathologic prognostic factors, including deoxyribonucleic acid content measured by means of flow cytometry, were analyzed for 282 patients. RESULTS: In 80% of the patients, the deoxyribonucleic acid patterns were nondiploid. In univariate analysis stage ( p p p = 0.01), and deoxyribonucleic acid ploidy ( p = 0.02) significantly predicted progression-free survival. In multivariate analysis stage ( p p = 0.001), deoxyribonucleic acid ploidy ( p = 0.02), and deoxyribonucleic acid index ( p = 0.02) retained independent prognostic value. Residual disease and deoxyribonucleic acid content retained independent prognostic value for stage III tumors but not for stage IV tumors. CONCLUSION: Deoxyribonucleic acid analysis with flow cytometry provides prognostic information about long-term progression-free survival from advanced ovarian carcinoma and should be considered in the stratification processes of patients in future clinical trials. This prognostic information appears to be inversely related to tumor burden. (Am J Obstet Gynecol 1996;175:1217-25.)

BACKGROUND The measurement of estrogen receptors (ER) in breast cancer specimens has traditionally been assessed with a dextran-coated charcoal assay (DCCA). More recently the immunohistochemical staining (IHC) method has gained increasing popularity because of its ability to use fixed tissue, assess needle biopsies, and reduce cost. Controversy exists over the accuracy of IHC compared with that of DCCA in determining ER. We compared these two techniques using tumor tissue obtained from a large group of females with lymph node positive breast carcinoma with long term follow-up. METHODS Breast carcinoma tissue was obtained from a large group of females with node positive breast carcinoma participating in two adjuvant chemotherapy trials. ER was determined by the traditional DCCA method and by IHC using the ER1D5 antibody. Disease free survival (DFS) and overall survival (OS) were assessed by each of these methods. RESULTS ER status was determined by DCCA and IHC in tumor tissue obtained from 316 females. A concordance of 79% was observed for the determination of ER-positive tumors. Of the discordant results, the majority of DCCA-negative, IHC-positive tumors could be explained by a low level of DCCA positivity (

Tissue factor pathway inhibitor (TFPI) is a novel agent that binds to tissue factor/VIIa complex and factor-Xa, thereby reducing the effect of tissue factor (TF) on inflammation and the extrinsic pathway of coagulation. We hypothesize that systemic treatment with TFPI may limit ischemia-reperfusion (IR) injury. Our experiment was designed to evaluate the effects of TFPI on IR in the spinal cord. Twenty-three adult New Zealand white rabbits had snare occlusion devices placed circumferentially around the aorta and tunneled to a subcutaneous position. Forty-eight hours later, in the fully awake state, the animals were treated with either TFPI (1 mg/kg bolus followed by a 1-hr infusion of 20 microgram/kg/min), or heparin (100 U/kg bolus) followed by a 1-hr infusion of 10 ml/kg/hr of PBS while controls received phosphate buffered saline (20 ml followed by a 1-hr infusion of 10 ml/kg/hr). The infrarenal aorta was occluded for 21 min in all groups via the snare device. Animals were observed for 3 days and neurologic recovery was graded by the Tarlov criteria. Results were evaluated as percent of animals with hindlimb recovery (Tarlov 3 and 4). At 24 hr postocclusion, 88% of the TFPI-treated animals had recovered neurologic function versus only 20% of heparin-treated animals and 10% of the phosphate buffered saline group (P=0.031 and 0.009, respectively). At 72 hr, 63% of the TFPI animals retained neurologic function versus 20% of heparin-treated animals and 10% of phosphate buffered saline-treated animals (P=0.032, TFPI versus phosphate buffered saline). The mechanism of action of TFPI is not completely understood, yet this drug may hold promise in the prevention of IR injury of the spinal cord.

Zebrafish are an excellent model for Duchenne muscular dystrophy. In particular, zebrafish provide a system for rapid, easy, and low-cost screening of small molecules that can ameliorate muscle damage in dystrophic larvae. Here we identify an optimal anti-sense morpholino cocktail that robustly knocks down zebrafish Dystrophin (dmd-MO). We use two approaches, muscle birefringence and muscle actin expression, to quantify muscle damage and show that the dmd-MO dystrophic phenotype closely resembles the zebrafish dmd mutant phenotype. We then show that the histone deacetylase (HDAC) inhibitor TSA, which has been shown to ameliorate the mdx mouse Duchenne model, can rescue muscle fiber damage in both dmd-MO and dmd mutant larvae. Our study identifies optimal morpholino and phenotypic scoring approaches for dystrophic zebrafish, further enhancing the zebrafish dmd model for rapid and cost-effective small molecule screening.

The notochord is a midline mesodermal structure with an essential patterning function in all vertebrate embryos. Zebrafish floating head (flh) mutants lack a notochord, but develop with prechordal plate and other mesodermal derivatives, indicating that flh functions specifically in notochord development. We show that floating head is the zebrafish homologue of Xnot, a homeobox gene expressed in the amphibian organizer and notochord. We propose that flh regulates notochord precursor cell fate.

Liver transplantation is now advocated for patients with early stage hepatocellular carcinoma (HCC) not amenable to surgical resection. In this article we review our experience with liver transplantation as treatment for patients with HCC and end-stage liver disease. Between April 1998 and May 2002, 36 patients with a diagnosis of HCC underwent liver transplantation at Ochsner Clinic Foundation. A retrospective analysis was performed examining pretransplant staging of disease, pathologic staging, disease recurrence, and patient survival. Cumulative 1- and 3-year patient survival rates are 80% and 61%, respectively. To date, none of our patients has developed evidence of recurrent cancer. Our data support liver transplantation as the treatment of choice for patients with unresectable early stage HCC.

Leucovorin, fluorouracil, and oxaliplatin (FOLFOX) is the standard adjuvant therapy for resected stage III colon cancer. Adding cetuximab to FOLFOX benefits patients with metastatic wild-type KRAS but not mutated KRAS colon cancer.To assess the potential benefit of cetuximab added to the modified sixth version of the FOLFOX regimen (mFOLFOX6) in patients with resected stage III wild-type KRAS colon cancer.A randomized trial of 2686 patients aged 18 years or older at multiple institutions across North America enrolled following resection and informed consent between February 10, 2004, and November 25, 2009. The primary randomized comparison was 12 biweekly cycles of mFOLFOX6 with and without cetuximab. KRAS mutation status was centrally determined. The trial was halted after a planned interim analysis of 48% of predicted events (246/515) occurring in 1863 (of 2070 planned) patients with tumors having wild-type KRAS. A total of 717 patients with mutated KRAS and 106 with indeterminate KRAS were accrued. The 2070 patients with wild-type KRAS provided 90% power to detect a hazard ratio (HR) of 1.33 (2-sided α = .05), with planned interim efficacy analyses after 25%, 50%, and 75% of expected relapses.Disease-free survival in patients with wild-type KRAS mutations. Secondary end points included overall survival and toxicity.Median (range) follow-up was 28 (0-68) months. The trial demonstrated no benefit when adding cetuximab. Three-year disease-free survival for mFOLFOX6 alone was 74.6% vs 71.5% with the addition of cetuximab (HR, 1.21; 95% CI, 0.98-1.49; P = .08) in patients with wild-type KRAS, and 67.1% vs 65.0% (HR, 1.12; 95% CI, 0.86-1.46; P = .38) in patients with mutated KRAS, with no significant benefit in any subgroups assessed. Among all patients, grade 3 or higher adverse events (72.5% vs 52.3%; odds ratio [OR], 2.4; 95% CI, 2.1-2.8; P.001) and failure to complete 12 cycles (33% vs 23%; OR, 1.6; 95% CI, 1.4-1.9; P.001) were significantly higher with cetuximab. Increased toxicity and greater detrimental differences in all outcomes were observed in patients aged 70 years or older.Among patients with stage III resected colon cancer, the use of cetuximab with adjuvant mFOLFOX6 compared with mFOLFOX6 alone did not result in improved disease-free survival.clinicaltrials.gov Identifier: NCT00079274.

Background. This study was designed to evaluate the prognostic importance of c-erbB2 overexpression in a standardized cohort of patients with axillary lymph node positive breast cancer. Methods. Paraffin embedded primary breast cancers from 354 patients with axillary lymph node positive breast cancer, treated on a North Central Cancer Treatment Group adjuvant protocol, were studied immunohistochemically. c-erbB2 staining was classified as negative, weak (1+), moderate (2+), or strong (3+) and was assessed for effectiveness as a predictor of outcome in univariate and Cox model multivariate analyses. Results. Twenty percent of specimens exhibited moderate or strong c-erbB2 staining. The median disease free survival period of the strong staining group was 2.9 years, compared with 7.1 years for all other patients (P = 0.01). The median overall survival for the strong staining group was 5 years, compared with 12 years for all other patients (P = 0.03). A definite correlation was noted between degree of nodal involvement and the likelihood of strong c-erbB2 staining (P = 0.001). There was also a significant correlation between c-erbB2 staining and higher nuclear grade and estrogen receptor negativity. In a multivariate analysis, c-erbB2 staining was not a significant predictor of either disease free survival or overall survival. Conclusion. According to this analysis, the strong correlation between c-erbB2 expression and degree of nodal involvement, higher grade disease, and estrogen receptor negativity suggests expression of this protooncogene product in a biologically more aggressive form of breast cancer. In a multivariate analysis, c-erbB2 expression was not an independent prognostic factor. Thus, c-erbB2 assessment did not appear to add significantly to the information provided by currently available standard disease parameters.

Nodular regenerative hyperplasia of the liver is an uncommon cause of portal hypertension. Patients with nodular regenerative hyperplasia have signs and symptoms of portal hypertension, without evidence of hepatocellular failure or encephalopathy. We report the case of a 44-year-old woman with recurrent esophageal bleeding and refractory ascites who had a history of hemosiderosis, hepatitis C, and chronic renal allograft rejection. Our preoperative diagnosis was cirrhotic end-stage liver disease and end-stage renal disease for which the patient underwent combined hepatic and renal transplantation. Her portal hypertension symptoms resolved, and her renal function has been normal for 18 months of follow-up. Histologic examination of the liver revealed nodular regenerative hyperplasia, and a review of the literature regarding the surgical management of patients with nodular regenerative hyperplasia revealed that various shunting procedures are generally recommended. After the failure of medical management in patients with nodular regenerative hyperplasia, portosystemic shunting may be indicated before proceeding to hepatic transplantation.

Background Patients with multiple liver metastases from colorectal cancer are at high risk of recurrence after resection. Hepatic artery infusion (HAI) alternating with systemic therapy after surgical resection may improve survival after surgery. Methods Patients with liver-only metastases from colorectal cancer amenable to resection/cryoablation were eligible. Previous adjuvant chemotherapy for a completely resected primary tumor was allowed. Alternating courses of HAI and systemic therapy included floxuridine (FUDR) by HAI. Systemic chemotherapy consisted of bolus leucovorin (LV) plus 5-fluorouracil (5-FU). Results Forty-nine patients had complete resection of their liver metastases, with 44% having more than 4 hepatic metastases and 78% having bilobar disease. Thirty-six patients had HAI FUDR alternating with systemic therapy. Patients received a median of 3.5 cycles (range, 1-4) and 3 cycles (range, 0-6) of therapy with HAI FUDR and systemic therapy, respectively. At the time of final analysis the estimated median disease-free survival and hepatic disease-free survival was 1.2 years (95% confidence interval [CI], 0.9-2.1) and 1.8 years (95% CI, 1.8-not available), respectively. Eleven patients (31%) were alive at this writing. All surviving patients had a minimum of 5.5 years of follow-up. Conclusion This trial of adjuvant chemotherapy in patients who underwent complete resection with unfavorable characteristics demonstrates apparent improvement in outcome compared with historical series treated with surgery alone. However the results of this trial and other randomized trials of HAI do not appear to support its use at this time because of the development of more effective systemic options.

Glomus tumors of the head and neck include those arising from the carotid body, jugular vein, and vagus nerve. Because these cannot be differentiated histologically, when encountering a large tumor mass involving more than one structure in the carotid sheath, one often cannot be sure whether the tumors are from one or more of these structures. The authors performed DNA flow cytometric analysis on a patient with a multicentric glomus tumor on the right side of the neck involving the carotid body, jugular vein, and vagus nerve, in an effort to determine the separate or similar origin of her tumor mass. Different DNA indices, including a double peak for the carotid body tumor, were obtained. There were three aneuploid tumors and one diploid tumor (DNA indices: carotid body 1.78, 2.04; jugular vein 2.20; vagus nerve 1.82). Different synthetic phase fractions were calculated for each aneuploid tumor except the second carotid body peak (carotid body 7.2; jugular vein 3.6; vagus nerve 4.8). The authors conclude that DNA flow cytometry may be useful in confirming the multicentric origin of tumors that encompass more than one histologically similar structure.

While hepatic resection of metastatic colorectal carcinoma is accepted as effective in selected patients, resection of metastases to other solid organs has not gained wide acceptance. We retrospectively reviewed the records of 49 patients who had resection of isolated pulmonary (18 patients) and hepatic (31 patients) metastases from the large bowel, comparing disease-free survival and overall survival. Tumor parameters analyzed included Dukes' stage, deoxyribonucleic acid (DNA) flow cytometry, and number of metastases. Dukes' B and diploid tumors had longer disease-free survival and overall survival than did Dukes' C and aneuploid tumors, though the difference was not significant. Patients with a single lung metastasis had a significantly longer disease-free survival (P = .02) than did patients with multiple metastases. Mean and median survival were longer in patients with lung metastases. Five-year actuarial survival was 19% for patients with liver metastases and 47% for patients with lung metastases. Resection of isolated pulmonary metastases from the large intestine results in survival comparable to or better than resection of hepatic metastases. An aggressive surgical approach is warranted in patients with isolated resectable pulmonary metastases of colorectal carcinoma.

In Xenopus, axis development is initiated by dorsally elevated levels of cytoplasmic β-catenin, an intracellular factor regulated by GSK3 kinase activity. Upon fertilization, factors that increase β-catenin stability are translocated to the prospective dorsal side of the embryo in a microtubule-dependent process. However, neither the identity of these factors nor the mechanism of their movement is understood. Here, we show that the GSK3 inhibitory protein GBP/Frat binds kinesin light chain (KLC), a component of the microtubule motor kinesin. Upon egg activation, GBP-GFP and KLC-GFP form particles and exhibit directed translocation. KLC, through a previously uncharacterized conserved domain, binds a region of GBP that is required for GBP translocation and for GSK3 binding, and competes with GSK3 for GBP. We propose a model in which conventional kinesin transports a GBP-containing complex to the future dorsal side, where GBP dissociates and contributes to the local stabilization of β-catenin by binding and inhibiting GSK3.

Background: This study evaluated the activity and tolerance for the combination of oral etoposide and paclitaxel as first-line therapy for patients with extensive SCLC. Methods: A total of 57 patients were enrolled in this study. A cycle of chemotherapy consisted of oral etoposide administered as 50mg BID on days 1 through 10 and paclitaxel administered as 150mg/m 2 IV (3h infusion) along with the first dose of etoposide on day 10. Patients were assessed for response to therapy (regression, stable disease, progression), survival, time to disease progression, and toxicity. Results/conclusions: Fifty-five patients were evaluable for efficacy parameters. Among the 55 patients, there were six with complete regression of disease, 18 with partial regression, 11 with regression, five with stable disease, and 15 with progressive disease, yielding an overall response rate of 63.6% (95% confidence interval, 50.0–76.0%). The 6-month and 1-year progression-free survival rates were 48.2 and 18.9%, respectively. The median time to disease progression was 5.8 months. The overall survival rates were 67.3% at 6 months and 41.8% at 1 year. The combination of oral etoposide and paclitaxel demonstrated significant efficacy as first-line therapy for extensive SCLC, with an overall response rate of 63.6% for 55 evaluable patients. In addition, the treatment was well tolerated with no unexpected toxicities.