Phase I study of emactuzumab single agent or in combination with paclitaxel in patients with advanced/metastatic solid tumors reveals depletion of immunosuppressive M2-like macrophages.

BACKGROUND: Emactuzumab is a monoclonal antibody against the colony-stimulating factor-1 receptor and targets tumor-associated macrophages (TAMs). This study assessed the safety, clinical activity, pharmacokinetics (PK) and pharmacodynamics (PD) of emactuzumab, as monotherapy and in combination with paclitaxel, in patients with advanced solid tumors. PATIENTS AND METHODS: This open-label, phase Ia/b study comprised two parts (dose escalation and dose expansion), each containing two arms (emactuzumab, every 2 or 3 weeks, as monotherapy or in combination with paclitaxel 80 mg/m2 weekly). The dose-escalation part explored the maximum tolerated dose and optimal biological dose (OBD). The dose-expansion part extended the safety assessment and investigated the objective response rate. A PK/PD analysis of serial blood, skin and tumor biopsies was used to explore proof of mechanism and confirm the OBD. RESULTS: No maximum tolerated dose was reached in either study arm, and the safety profile of emactuzumab alone and in combination does not appear to preclude its use. No patients receiving emactuzumab monotherapy showed an objective response; the objective response rate for emactuzumab in combination with paclitaxel was 7% across all doses. Skin macrophages rather than peripheral blood monocytes or circulating colony-stimulating factor-1 were identified as an optimal surrogate PD marker to select the OBD. Emactuzumab treatment alone and in combination with paclitaxel resulted in a plateau of immunosuppressive TAM reduction at the OBD of 1000 mg administered every 2 weeks. CONCLUSIONS: Emactuzumab showed specific reduction of immunosuppressive TAMs at the OBD in both treatment arms but did not result in clinically relevant antitumor activity alone or in combination with paclitaxel. (ClinicalTrials.gov Identifier: NCT01494688).

Hot topic: performance of bovine high-density genotyping platforms in Holsteins and Jerseys.

Two high-density single nucleotide polymorphism (SNP) genotyping arrays have recently become available for bovine genomic analyses, the Illumina High-Density Bovine BeadChip Array (777,962 SNP) and the Affymetrix Axiom Genome-Wide BOS 1 Array (648,874 SNP). These products each have unique design and chemistry attributes, and the extent of marker overlap and their potential utility for quantitative trait loci fine mapping, detection of copy number variation, and multibreed genomic selection are of significant interest to the cattle community. This is the first study to compare the performance of these 2 arrays. Deoxyribonucleic acid samples from 16 dairy cattle (10 Holstein, 6 Jersey) were used for the comparison. An independent set of DNA samples taken from 46 Jersey cattle and 18 Holstein cattle were used to ascertain the amount of SNP variation accounted by the 16 experimental samples. Data were analyzed with SVS7 software (Golden Helix Inc., Bozeman, MT) to remove SNP having a call rate less than 90%, and linkage disequilibrium pruning was used to remove linked SNP (r² ≥ 0.9). Maximum, average, and median gaps were calculated for each analysis based on genomic position of SNP on the bovine UMD3.1 genome assembly. All samples were successfully genotyped (≥ 98% SNP genotyped) with both platforms. The average number of genotyped SNP in the Illumina platform was 775,681 and 637,249 for the Affymetrix platform. Based on genomic position, a total of 107,896 SNP were shared between the 2 platforms; however, based on genotype concordance, only 96,031 SNP had complete concordance at these loci. Both Affymetrix BOS 1 and Illumina BovineHD genotyping platforms are well designed and provide high-quality genotypes and similar coverage of informative SNP. Despite fewer total SNP on BOS 1, 19% more SNP remained after linkage disequilibrium pruning, resulting in a smaller gap size (5.2 vs. 6.9 kb) in Holstein and Jersey samples relative to BovineHD. However, only 224,115 Illumina and 241,038 Affymetrix SNP remained following removal of SNP with a minor allele frequency of zero in Holstein and Jersey samples, resulting in an average gap size of 11,887 bp and 11,018 bp, respectively. Combining the 354,348 informative (r² ≥ 0.9), polymorphic (minor allele frequency ≥ 0), unique SNP data from both platforms decreased the average gap size to 7,560 bp. Genome-wide copy number variant analyses were performed using intensity files from both platforms. The BovineHD platform provided an advantage to the copy number variant data compared with the BOS 1 because of the larger number of SNP, higher intensity signals, and lower background effects. The combined use of both platforms significantly improved coverage over either platform alone and decreased the gap size between SNP, providing a valuable tool for fine mapping quantitative trait loci and multibreed animal evaluation.

Microtubules remodel actomyosin networks in Xenopus egg extracts via two mechanisms of F-actin transport.

Interactions between microtubules and filamentous actin (F-actin) are crucial for many cellular processes, including cell locomotion and cytokinesis, but are poorly understood. To define the basic principles governing microtubule/F-actin interactions, we used dual-wavelength digital fluorescence and fluorescent speckle microscopy to analyze microtubules and F-actin labeled with spectrally distinct fluorophores in interphase Xenopus egg extracts. In the absence of microtubules, networks of F-actin bundles zippered together or exhibited serpentine gliding along the coverslip. When microtubules were nucleated from Xenopus sperm centrosomes, they were released and translocated away from the aster center. In the presence of microtubules, F-actin exhibited two distinct, microtubule-dependent motilities: rapid ( approximately 250-300 nm/s) jerking and slow ( approximately 50 nm/s), straight gliding. Microtubules remodeled the F-actin network, as F-actin jerking caused centrifugal clearing of F-actin from around aster centers. F-actin jerking occurred when F-actin bound to motile microtubules powered by cytoplasmic dynein. F-actin straight gliding occurred when F-actin bundles translocated along the microtubule lattice. These interactions required Xenopus cytosolic factors. Localization of myosin-II to F-actin suggested it may power F-actin zippering, while localization of myosin-V on microtubules suggested it could mediate interactions between microtubules and F-actin. We examine current models for cytokinesis and cell motility in light of these findings.

An in vivo animal model of gene therapy for leukocyte adhesion deficiency.

Leukocyte adhesion deficiency (LAD) is an inherited disorder of leukocyte function that is caused by defects in the CD18 gene and is associated with diminished cell surface expression of CD11/CD18 proteins. We have developed an in vivo model for gene therapy of LAD. Recombinant retroviruses were used to transduce a functional human CD18 gene into murine bone marrow cells which were transplanted into lethally irradiated syngeneic recipients. A reliable flow cytometric assay for human CD18 in transplant recipients was developed based on: (a) the availability of human specific CD18 monoclonal antibodies and (b) the observation that human CD18 can form chimeric heterodimers with murine CD11a on the cell surface. Human CD18 was detected on leukocytes in a substantial number of transplant recipients for at least 6 mo suggesting that the gene had been transduced into stem cells. Expression was demonstrated in several lineages of a variety of hematopoietic tissues, but was consistently highest and most frequent in granulocytes. Murine granulocytes demonstrated appropriate posttranscriptional regulation of human CD18 in response to activation of protein kinase C. No apparent untoward effects of human CD18 expression were noted in transplant recipients. These studies suggest a specific strategy for LAD gene therapy that may be effective and safe.

Relationship between Liver Mitochondrial Respiration and Proton Leak in Low and High RFI Steers from Two Lineages of RFI Angus Bulls.

The objective of this research is to evaluate liver mitochondrial oxygen consumption and proton leak kinetics in progeny from two lineages of Angus bulls with high and low residual feed intake (RFI). Two Angus bulls were selected based on results from a genetic test for RFI and were used as sires. Eight offspring at 10-11 months of age from each sire were housed in individual pens for 70-105 days following a diet adaptation period of 14 days. Progeny of the low RFI sire had 0.57 kg/d (P = 0.05) lower average RFI than progeny of the high RFI sire. There was no difference in dry matter intake between low and high RFI steers, but low RFI steers gained more body weight (P = 0.02) and tended to have higher average daily gains (P = 0.07). State 3 and State 4 respiration, RCR, and proton leak did not differ between high and low RFI steers (P = 0.96, P = 0.81, P = 0.93, and P = 0.88, resp.). Therefore, the increase in bodyweight gain which distinguished the low RFI steers from the high RFI steers may be associated with other metabolic mechanisms that are not associated with liver mitochondrial respiration and proton leak kinetics.

Rapid acquisition of unknown DNA sequence adjacent to a known segment by multiplex restriction site PCR.

The determination of unknown DNA sequences around a known locus has important applications in molecular genetics, specifically in genomic walking and genome mapping. Several PCR-based methods have been reported to address this issue, but they often involve multiple, time-consuming steps. We have previously described a technique known as restriction site PCR (RS-PCR) that allows sequence acquisition faster than the existing methods. The method involves PCR using four separate universal primers that are representative of given restriction enzyme sites (RS primers), and a specific primer from one end of the known sequence. We have now significantly improved the technique by mixing the four universal primers into one PCR tube with the first specific primer. This is followed by a nested PCR with the mixed RS primers and an internal specific primer, after which the product is sequenced by direct automated sequencing. The technique, called multiplex RS-PCR (mRS-PCR), is reproducible and can be used to obtain unknown sequence adjacent to known sequences in both the upstream and downstream directions. We illustrate the application of mRS-PCR in the acquisition of approximately 780 bp of genomic sequence starting from a known sequence of approximately 120 bp. Multiplex RS-PCR appears to be the fastest of all methods that address the issue of unknown sequence retrieval adjacent to a known region.

Dependence of fluorodeoxyuridine-induced cytotoxicity and megabase DNA fragment formation on S phase progression in HT29 cells.

The relationship between cell cycle progression and induction of DNA double-strand breaks and cytotoxicity by exposure to fluorodeoxyuridine (FdUrd) was studied in HT29 human colon cancer cells. Fractionation of drug-treated populations by centrifugal elutriation yielded subpopulations having widely divergent abilities to progress through S phase in the presence of the drug. One of these subpopulations, which appeared to undergo coordinated growth arrest, was resistant to FdUrd cytotoxicity and DNA damage. In contrast, the subpopulation which was able to progress furthest through S phase in the presence of FdUrd underwent unbalanced growth arrest (i.e., increase in size and mass out of proportion to DNA synthesis), and displayed both DNA double-strand break formation (assayed by pulsed field gel electrophoresis) and loss of clonogenicity. When cells were elutriated prior to drug treatment, producing fractions enriched in cells at various cell cycle stages, no significant differences in sensitivity to FdUrd-induced cytotoxicity were detected among elutriation fractions. These findings support the model that, in HT29 cells, progression into and through S phase during drug treatment is an important determinant of FdUrd-induced DNA damage and cytotoxicity, but that the cell cycle position at the start of drug exposure is not a critical factor for these effects.

Selective differential fingerprinting. A method for identifying differentially expressed genes in a family between two samples.

A method termed selective differential fingerprinting (SDF) has been developed that enables one to investigate the level of expression for a family of genes between two samples. SDF produces a fingerprint (on a sequencing gel) on reverse transcription polymerase chain reaction (RT-PCR) of a sample with degenerate primers designed from conserved regions of a family of genes. By comparing fingerprints obtained after SDF with primers representing the transforming growth factor-beta (TGF beta) family of growth factors between a low-grade and a high-grade tumor from the same patient, a TGF beta family member known as osteogenic protein 1 (OP-1) or bone morphogenic protein 7 (BMP-7) was found to be greatly overexpressed in the high-grade tumor compared to the low-grade one. SDF also has the potential to identify novel genes. SDF offers a general way to identify differentially expressed genes for a family between two given samples.

Evidence for the upregulation of osteogenic protein-1 mRNA expression in musculoskeletal neoplasms.

Little is known about bone and cartilage tumors at the molecular level; thus, the identification of genes associated with these tumors may be useful as markers and therapeutic targets. To address this issue and to test the hypothesis that abnormal expression of one or more growth factors in the transforming growth factor-beta superfamily is associated with musculoskeletal neoplasia, degenerate primers based on the conserved sequences in these genes were made for screening tumor samples by reverse transcription-polymerase chain reaction. First, these primers were used to obtain a comparative profile between a low-grade chondrosarcoma and its dedifferentiated high-grade counterpart in the same patient. This experiment identified an amplified DNA product in the high-grade sample that was identical to osteogenic protein-1/bone morphogenetic protein-7. Osteogenic protein-1 mRNA expression was 17-fold greater in this high-grade sample than in the low-grade one. Osteogenic protein-1 was highly expressed (three of three) in human osteosarcoma cell lines but was not expressed (zero of four) in normal osteoblast samples. Screening for gene expression of osteogenic protein-1 in 57 osteosarcomas and chondrosarcomas indicated that 44% (range: 38-52%) of them were positive for osteogenic protein-1 mRNA. Screening of breast and prostate tumors revealed a similar association with osteogenic protein-1 mRNA expression.

Osteoid osteoma of the elbow: a diagnostic challenge.

BACKGROUND: Osteoid osteoma is a painful benign neoplasm that is rarely found in the elbow region. METHODS: The study included fourteen patients, and we believe that this is the largest reported series of patients with osteoid osteoma of the elbow evaluated at one institution. Most of the patients had had symptoms for a prolonged period and had had multiple invasive procedures before an accurate diagnosis was made. Although findings on physical examination generally are nonspecific and are not always accurate in localizing the lesion, plain tomograms and computed tomography scans were most helpful in identifying the nidus in the present study. Thirteen of the patients had limited motion of the elbow before the definitive diagnosis was made, and ten of these thirteen had a mean flexion contracture of 38 degrees. RESULTS: Removal of the nidus resulted in relief of pain and improvement in the range of motion of the elbow in all fourteen patients. A persistent postoperative flexion contracture was more common in the patients who had had a previous arthrotomy of the elbow than in those who had not had that procedure. CONCLUSIONS: It is important to recognize this uncommon entity to avoid the morbidity associated with a prolonged delay in diagnosis. Because the symptoms resolve after excision of the lesion, the surgeon can avoid unnecessary soft-tissue dissection and release of the contracture.

Revision of a failed cemented total hip prosthesis with insertion of an acetabular component without cement and a femoral component with cement. A five to eight-year follow-up study.

Sixty-one consecutive so-called hybrid revision total hip arthroplasties were performed in fifty-five patients by one surgeon, from 1986 through 1988, for mechanical failure of a cemented total hip prosthesis. In all of the patients, the acetabular and femoral components were revised to a porous-coated Harris-Galante acetabular component inserted without cement and an Iowa femoral component inserted with cement. Contemporary cementing techniques were used, but structural bone graft was not. The over-all prevalence of repeat revision for aseptic loosening was 0 per cent for the acetabular components and 3 per cent (two hips) for the femoral components. In addition, 2 per cent (one) of the acetabular components and 5 per cent (three) of the femoral components demonstrated radiographic evidence of loosening. In the forty-three patients (forty-nine hips) who were alive at an average of seventy-four months (range, sixty to ninety-five months) after the revision, none of the acetabular components and 2 per cent (one) of the femoral components were revised again for aseptic loosening. An additional 2 per cent (one) of the acetabular components and 6 per cent (three) of the femoral components were radiographically loose. Ninety-eight per cent (forty-one) of the forty-two living patients (98 per cent [forty-seven] of the forty-eight hips) who had a clinical examination at least five years after the revision had increased function; 90 per cent (thirty-eight) of these patients (forty-four [92 per cent] of the hips) were satisfied with the result. The group that had a hybrid revision was compared with a group of seventy patients (seventy-four hips) who had had a revision total hip arthroplasty with use of contemporary cementing techniques for both components. These revisions had been performed by the same surgeon, before he performed the hybrid revisions, and the prevalence of repeat revision of the acetabular component was 7 per cent (five hips) and that of the femoral component was 4 per cent (three hips). In addition, 16 per cent (twelve) of the acetabular components and 3 per cent (two) of the femoral components were radiographically loose. The comparison group was not a consecutive series, as only the patients who had had radiographs made five to eight years after the revision were evaluated. In the fifty-two such patients (fifty-six hips) who were alive at five years after the revision with cement (average duration of radiographic follow-up, seventy-seven months; range, sixty to ninety-nine months), 9 per cent (five) of the acetabular components and 5 per cent (three) of the femoral components were revised again for aseptic loosening. An additional 21 per cent (twelve) of the acetabular components and 4 per cent (two) of the femoral components were radiographically loose. The results of the present study demonstrated a significant improvement (p = 0.0001) in the survival of the acetabular component of so-called hybrid revision total hip arthroplasties compared with that of revision total hip arthroplasties with cement performed by the same surgeon and followed for a comparable period.

Chondrosarcoma of the pelvis. A review of sixty-four cases.

BACKGROUND: Treatment of pelvic chondrosarcoma is a difficult problem for the musculoskeletal oncologist. Poor rates of survival and high rates of local recurrence after surgical treatment have been reported in previous studies. The present study was designed to review the long-term oncologic and functional outcomes of surgical management in a large series of patients with pelvic chondrosarcoma who were treated at a single institution. METHODS: The cases of sixty-four patients with localized pelvic chondrosarcoma that had been surgically treated between 1975 and 1996 were reviewed retrospectively. The study was limited to patients who had received no previous treatment for chondrosarcoma. There were forty-one male and twenty-three female patients who had a mean age of forty-seven years (range, fifteen to eighty-eight years). The patients were followed for a minimum of three years or until death. The median duration of follow-up of the living patients was 140 months (range, thirty-nine to 295 months). RESULTS: Thirty-three of the sixty-four patients were first seen with grade-1 chondrosarcoma; twenty-three, with grade-2; one, with grade-3; and seven, with grade-4 (dedifferentiated chondrosarcoma). Thirteen patients had a hemipelvectomy to achieve local tumor control, whereas fifty-one patients underwent a limb-salvage procedure. Twelve patients (19%) had local recurrence, and eleven (17%) had distant metastases. At the time of the final follow-up, forty-four patients (69%) were alive without evidence of disease, thirteen (20%) had died of the disease, six (9%) had died of unrelated causes, and one (2%) was alive with disease. Less than a wide surgical margin correlated with local recurrence (p = 0.014). High-grade tumors correlated with poor overall survival (p < 0.001). All patients who had a limb-salvage procedure were able to walk at the time of the final follow-up, and they had a mean functional score of 77%, according to the system of the Musculoskeletal Tumor Society. CONCLUSIONS: Aggressive surgical resection of pelvic chondrosarcoma results in long-term survival of the majority of patients. There is a high correlation between tumor grade and overall or disease-free survival.

Evaluation of bull prolificacy on commercial beef cattle ranches using DNA paternity analysis.

SNP-based DNA testing was used to assign paternity to 5,052 calves conceived in natural service multisire breeding pastures from 3 commercial ranches in northern California representing 15 calf crops over 3 yr. Bulls present for 60 to 120 d at a 25:1 cow to bull ratio in both fall and spring breeding seasons in ∼40 ha or smaller fenced breeding pastures sired a highly variable (P < 0.001) number of calves (Ncalf), ranging from 0 (4.4% of bulls present in any given breeding season) to 64 calves per bull per breeding season, with an average of 18.9 ± 13.1. There was little variation in Ncalf among ranches (P = 0.90), years (P = 0.96), and seasons (P = 0.94). Bulls varied widely (P < 0.01) in the average individual 205-d adjusted weaning weight (I205) of progeny, and I205 varied between years (P < 0.01) and seasons (P < 0.01) but not ranches (P = 0.29). The pattern for cumulative total 205-d adjusted weaning weight of all progeny sired by a bull (T205) was highly correlated to Ncalf, with small differences between ranches (P = 0.35), years (P = 0.66), and seasons (P = 0.20) but large differences (P < 0.01) between bulls, ranging from an average of 676 to 8,838 kg per bull per calf crop. The peak Ncalf occurred at about 5 yr of age for bulls ranging from 2 to 11 yr of age. Weekly conception rates as assessed by date of calving varied significantly and peaked at wk 3 of the calving season. The distribution of calves born early in the calving season was disproportionately skewed toward the highly prolific bulls. The DNA paternity testing of the subset of those calves born in wk 3 of the calving season was highly predictive of overall bull prolificacy and may offer a reduced-cost DNA-based option for assessing prolificacy. Prolificacy of young bulls in their first breeding season was positively linearly related (P < 0.05) to subsequent breeding seasons, explaining about 20% of the subsequent variation. Prolificacy was also positively linearly related (P < 0.05) to scrotal circumference (SC) EPD for Angus bulls that had SC EPD Beef Improvement Federation accuracies greater than 0.05. Varying prolificacy of herd bulls has implications for the genetic composition of replacement heifers, with the genetics of those bulls siring an increased number of calves being disproportionately represented in the early-born replacement heifer pool.

The accuracies of DNA-based estimates of genetic merit derived from Angus or multibreed beef cattle training populations.

Several organizations have developed prediction models for molecular breeding values (MBV) for quantitative growth and carcass traits in beef cattle using Bovine SNP50 genotypes and phenotypic or EBV data. Molecular breeding values for Angus cattle have been developed by IGENITY, Pfizer Animal Genetics, and a collaboration between researchers from Iowa State University and the University of Missouri-Columbia (ISU/UMC). The U.S. Meat Animal Research Center (USMARC; Clay Center, NE) has also developed MBV for 16 cattle breeds using 2 multibreed populations, the Germplasm Evaluation (GPE) Program and the 2,000 Bull Project (2K(ALL)), and 2 single breed subpopulations of the 2,000 Bull Project, Angus (2K(AN)) and Hereford (2K(HH)). In this study, these MBV were assessed relative to commercial ranch EBV estimated from the progeny phenotypes of Angus bulls naturally mated in multisire breeding pastures to commercial cows: 121 for USMARC MBV, 99 for ISU/UMC MBV, and 29 for IGENITY and Pfizer MBV (selected based on number of progeny carcass records). Five traits were analyzed: weaning weight (WW), HCW, marbling score (MS), rib-eye muscle area (RE), and, for IGENITY and Pfizer only, feedlot ADG. The average accuracies of MBV across traits were 0.38 ± 0.05 for IGENITY, 0.61 ± 0.12 for Pfizer, 0.46 ± 0.12 for ISU/UMC, 0.16 ± 0.04 for GPE, 0.26 ± 0.05 for 2K(ALL), 0.24 ± 0.04 for 2K(AN), and 0.02 ± 0.12 for 2K(HH). Angus-based MBV (IGENITY, Pfizer, ISU/UMC, and 2K(AN)) explained larger proportions of genetic variance in this population than GPE, 2K(ALL), or 2K(HH) MBV for the same traits. In this data set, IGENITY, Pfizer, and ISU/UMC MBV were predictive of realized performance of progeny, and incorporation of that information into national genetic evaluations would be expected to improve EPD accuracy, particularly for young animals.

Accuracy of genomic breeding values in multibreed beef cattle populations derived from deregressed breeding values and phenotypes.

Genomic selection involves the assessment of genetic merit through prediction equations that allocate genetic variation with dense marker genotypes. It has the potential to provide accurate breeding values for selection candidates at an early age and facilitate selection for expensive or difficult to measure traits. Accurate across-breed prediction would allow genomic selection to be applied on a larger scale in the beef industry, but the limited availability of large populations for the development of prediction equations has delayed researchers from providing genomic predictions that are accurate across multiple beef breeds. In this study, the accuracy of genomic predictions for 6 growth and carcass traits were derived and evaluated using 2 multibreed beef cattle populations: 3,358 crossbred cattle of the U.S. Meat Animal Research Center Germplasm Evaluation Program (USMARC_GPE) and 1,834 high accuracy bull sires of the 2,000 Bull Project (2000_BULL) representing influential breeds in the U.S. beef cattle industry. The 2000_BULL EPD were deregressed, scaled, and weighted to adjust for between- and within-breed heterogeneous variance before use in training and validation. Molecular breeding values (MBV) trained in each multibreed population and in Angus and Hereford purebred sires of 2000_BULL were derived using the GenSel BayesCπ function (Fernando and Garrick, 2009) and cross-validated. Less than 10% of large effect loci were shared between prediction equations trained on (USMARC_GPE) relative to 2000_BULL although locus effects were moderately to highly correlated for most traits and the traits themselves were highly correlated between populations. Prediction of MBV accuracy was low and variable between populations. For growth traits, MBV accounted for up to 18% of genetic variation in a pooled, multibreed analysis and up to 28% in single breeds. For carcass traits, MBV explained up to 8% of genetic variation in a pooled, multibreed analysis and up to 42% in single breeds. Prediction equations trained in multibreed populations were more accurate for Angus and Hereford subpopulations because those were the breeds most highly represented in the training populations. Accuracies were less for prediction equations trained in a single breed due to the smaller number of records derived from a single breed in the training populations.

Ewing's sarcoma: presentation and management.

Great strides have been made in the diagnosis and treatment of patients with Ewing's sarcoma. With the advent of modern chemotherapy, the long-term survival has improved to approximately 70%. Standard treatment for local control of the primary lesion has, historically, been chemotherapy and radiation. Currently, surgical resection has become a more effective option in the multidisciplinary treatment of patients with this disease. These current concepts and developments in the presentation and management of Ewing's sarcoma are discussed.

Sacropelvic resection for recurrent anorectal cancer. A multidisciplinary approach.

A multimodal approach including preoperative external beam radiation, surgical resection, and intraoperative electron radiation was used in 23 patients with locally advanced anal or recurrent rectal cancers involving the sacrum. The proximal extent of complete sacral resection was S2 in three patients, S3 in 12 patients, S4 in two patients, and S5 in one patient. The tumor was confined to the anterior sacral cortex in five patients. The resection was marginal in 10, contaminated marginal in 11, and intralesional in two patients. At 19 to 54 months of followup, five patients are alive without evidence of disease and four are alive with disease. Twelve patients died of their disease, and two died of other causes. There was a mean survival of 32.9 months for the patients who were alive at followup. Kaplan-Meier survival for all patients was 82% at 1 year and 73% at 2 years, with death of disease as an endpoint. Thirteen (57%) patients had another local recurrence develop at a mean of 17.2 months. Eight (35%) patients had metastatic disease develop at a mean of 16.3 months. Proper patients selection is important in ensuring a favorable outcome from this aggressive surgery.

Reoperation for failed prosthetic replacement used for limb salvage.

Patients with segmental bone and joint replacement prostheses because of tumors increasingly need revision surgery because of their long term survival. Between 1970 and 1990, 208 custom prosthetic replacements were performed for limb salvage in patients with tumors. Reoperations were required in 52 patients. The mean time to reoperation was 37 months. The reoperation procedures included 35 prosthetic revisions, 11 amputations, four arthrodeses, one vascularized fibular graft, and one open reduction and internal fixation of a fracture with supplemental bone graft. Functional assessment using the new Musculoskeletal Tumor Society scoring system was available for the 36 living patients, and their mean rating was 63% (18.9) at 12 years' mean followup. Of the 35 patients who received a new prosthesis, 12 (33%) patients needed a third operation at mean followup of 68 months. The probability of prosthetic survival in the group of 35 patients needing revision to the same or another prosthesis was 79% at 5 years and 65% at 10 years. The chance and frequency of needing reoperation increased as patients survived longer. Reoperations for tumor recurrence or infection usually resulted in amputation. Reoperation for failed initial segmental bone and joint prosthetic replacement is feasible and effective and can be done without jeopardizing subsequent patient and implant survival or without significantly affecting functional results compared with the values before reoperation.

Direct observation of microtubule-f-actin interaction in cell free lysates.

Coordinated interplay of the microtubule and actin cytoskeletons has long been known to be crucial for many cellular processes including cell migration and cytokinesis. However, interactions between these two systems have been difficult to document by conventional approaches, for a variety of technical reasons. Here the distribution of f-actin and microtubules were analyzed in the absence of fixation using Xenopus egg extracts as an in vitro source of microtubules and f-actin, demembranated Xenopus sperm to nucleate microtubule asters, fluorescent phalloidin as a probe for f-actin, and fluorescent tubulin as a probe for microtubules. F-actin consistently colocalized in a lengthwise manner with microtubules of asters subjected to extensive washing in flow chambers. F-actin-microtubule association was heterogenous within a given aster, such that f-actin is most abundant toward the distal (plus) ends of microtubules, and microtubules heavily labeled with f-actin are found in close proximity to microtubules devoid of f-actin. However, this distribution changed over time, in that 5 minute asters had more f-actin in their interiors than did 15 minute asters. Microtubule association with f-actin was correlated with microtubule bending and kinking, while elimination of f-actin resulted in straighter microtubules, indicating that the in vitro interaction between f-actin and microtubules is functionally significant. F-actin was also found to associate in a lengthwise fashion with microtubules in asters centrifuged through 30% sucrose, and microtubules alone (i.e. microtubules not seeded from demembranated sperm) centrifuged through sucrose, indicating that the association cannot be explained by flow-induced trapping and alignment of f-actin by aster microtubules. Further, cosedimentation analysis revealed that microtubule-f-actin association could be reconstituted from microtubules assembled from purified brain tubulin and f-actin assembled from purified muscle actin in the presence, but not the absence, of Xenopus oocyte microtubule binding proteins. The results provide direct evidence for an association between microtubules and f-actin in vitro, indicate that this interaction is mediated by one or more microtubule binding proteins, and suggest that this interaction may be responsible for the mutual regulation of the microtubule and actomyosin cytoskeletons observed in vivo.