Frederick Singer - Academia.edu (original) (raw)

Papers by Frederick Singer

Journal of submicroscopic cytology and pathology, 1998

In Paget's disease of bone, osteoclasts are increased in number and size and contain intracel... more In Paget's disease of bone, osteoclasts are increased in number and size and contain intracellular paramyxoviral-like inclusions which cross-react with antibody against measles, respiratory syncytial, and canine distemper viral nucleocapsid antigens. Moreover, measles virus nucleocapsid transcripts are present in pagetic osteoclasts and their mononuclear precursors formed in vitro. The present study was undertaken to morphologically assess pagetic osteoclasts formed in culture; special attention has been directed towards the ultrastructural identification of nuclear and cytoplasmic inclusions. Pagetic osteoclasts were produced in long-term cultures of non-adherent bone marrow mononuclear cells derived from involved bone of patients with Paget's disease. These cultured osteoclasts had many of the ultrastructural features of pagetic osteoclasts in vivo. Of interest, no viral-like inclusions were observed in either the multinucleated osteoclasts or mononuclear cell precursors i...

Cancer research, Jan 15, 2003

Detection of genetic markers associated with early breast cancer may prove clinically relevant fo... more Detection of genetic markers associated with early breast cancer may prove clinically relevant for identifying patients at increased risk for relapse. Loss of heterozygosity (LOH), a specific genetic aberration, commonly occurs during breast cancer initiation and metastasis. Early detection of tumor metastasis to bone marrow (BM) using conventional histochemical techniques has been limited because of suboptimal efficiency and sensitivity. Because bone is such a common site of breast cancer recurrence, we sought to determine whether microsatellite markers associated with breast cancer could be detected in BM aspirates from patients with early-stage breast cancer. Cell-free plasma from BM aspirates in 48 patients was assessed for LOH using a panel of eight polymorphic microsatellite markers. LOH was detected in 11 (23%) of 48 patients' BM aspirates. Advancing American Joint Committee on Cancer (AJCC) stage was associated with an increased incidence of LOH. Concordance was present ...

Orphanet journal of rare diseases, Jan 24, 2012

Fibrous dysplasia (FD) is sometimes accompanied by extraskeletal manifestations that can include ... more Fibrous dysplasia (FD) is sometimes accompanied by extraskeletal manifestations that can include any combination of café-au-lait macules, hyperfunctioning endocrinopathies, such as gonadotropin-independent precocious puberty, hyperthyroidism, growth hormone excess, FGF23-mediated renal phosphate wasting, and/or Cushing syndrome, as well as other less common features. The combination of any of these findings, with or without FD, is known as McCune-Albright syndrome (MAS). The broad spectrum of involved tissues and the unpredictable combination of findings owe to the fact that molecular defect is due to dominant activating mutations in the widely expressed signaling protein, Gsα, and the fact these mutations arises sporadically, often times early in development, prior to gastrulation, and can distribute across many or few tissues.The complexity can be mastered by a systematic screening of potentially involved tissues and cognizance that the pattern of involved tissues is established, ...

The Journal of Clinical Endocrinology & Metabolism, 2007

Context: Retention of technetium-99m-sestamibi (99mTc-sestamibi) by parathyroid adenomas appears ... more Context: Retention of technetium-99m-sestamibi (99mTc-sestamibi) by parathyroid adenomas appears to be due to the loss of at least one membrane transporter, multidrug resistance 1 (MDR1), and possibly another, multidrug resistance-associated protein 1 (MRP1). Objective: The objective was to determine whether hypermethylation of either gene plays a role in their expression and 99mTc-sestamibi retention. Design: This was a retrospective study on a convenience sample of paraffin-embedded parathyroid glands. Setting: The study was performed at the John Wayne Cancer Institute at Saint John’s Health Center (Santa Monica, CA). Patients: Forty-eight patients with primary hyperparathyroidism and five patients without parathyroid disease undergoing thyroid surgery provided 27 adenomatous, 10 hyperplastic, and 16 normal parathyroid glands. Intervention: We performed immunohistochemistry, real-time quantitative RT-PCR, and methylation-specific PCR for MDR1 and MRP1 on archival parathyroid tissu...

The Journal of Clinical Endocrinology & Metabolism, 2005

We agree that geometric mean would be a more appropriate way to provide a statistical summary of ... more We agree that geometric mean would be a more appropriate way to provide a statistical summary of the parathyroid weights but chose the mean weight as had been reported in the five autopsy series of the weights of normal parathyroid glands. As Dr. Parfitt has indicated, this does not affect our conclusion that the weight alone of a parathyroid gland is not sufficient to determine whether a gland is normal. With respect to whether dehydration might account for the differences in gland weights between autopsy and surgical series, we do not know the answer. We have not noted evidence of dehydration in parathyroid glands at autopsy. In more than 3000 cases at the Massachusetts General Hospital, the incidence of double adenomas of the parathyroid was 1.2%, and in the next largest series at the Mayo Clinic it was 1.9% (3). Nevertheless it is possible that very small microadenomas might be detected in higher numbers if serial sections of apparently normal glands were studied. We doubt this would explain our results but will seek approval of our Institutional Review Board to examine the largest normal glands in our series.

Clinical Chemistry, 2009

BACKGROUND: Prostate cancer (PCa) detection using serum-based prostate specific antigen (PSA) is ... more BACKGROUND: Prostate cancer (PCa) detection using serum-based prostate specific antigen (PSA) is limited by frequent false-positive and-negative results. Genetic aberrations such as allelic imbalance (AI) and epigenetic changes such as promoter hypermethylation have been detected in circulating DNA of cancer patients. We hypothesized that circulating multimarker DNA assays detecting both genetic and epigenetic markers in serum would be useful in assessing PCa patients. METHODS: We assayed blood from healthy male donors (n ϭ 40) and 83 patients with American Joint Cancer Committee (AJCC) stage I-IV PCa. DNA was assayed for AI of 6 genome microsatellites. We assessed methylation of RASSF1, RARB2, and GSTP1 using a methylation-specific PCR assay and analyzed the sensitivity of each assay for the detection of genetic or epigenetic changes in circulating DNA. The relation between circulating tumor-related DNA detection and prognostic factors was investigated. RESULTS: The proportion of patients demonstrating AI for Ն1 marker was 47% (38 of 81 patients). Methylation biomarkers were detected in 24 of 83 patients (28%). By combining 2 DNA assays, the number of PCa patients positive for Ն1 methylated or LOH marker increased (52 of 83; 63%). The combined assays detected PCa in 15 of 24 patients (63%) with normal PSA concentrations. The combination of the DNA assays detected the presence of PCa regardless of AJCC stage or PSA concentration. Combination of the DNA and PSA assays gave 89% sensitivity. CONCLUSIONS: This pilot study demonstrates that the combined circulating DNA multimarker assay identifies patients with PCa and may yield information independent of AJCC stage or PSA concentration.

Annals of Surgical Oncology, 2010

The purpose of this study was to report our experience with sentinel lymph node dissection (SLND)... more The purpose of this study was to report our experience with sentinel lymph node dissection (SLND) for papillary thyroid carcinoma, to evaluate the feasibility and safety of the procedure, and to examine its potential utility as a guide for central neck dissection. A retrospective chart review of patients undergoing total thyroidectomy from January 1998 thru January 2010 was conducted. Intratumoral injection of blue dye was used to identify the SLN. Central neck dissection (CND) was performed if the SLN was positive on frozen section. Locally advanced disease, previous thyroid surgery, or lymphadenopathy on preoperative imaging or intraoperative palpation were exclusion criteria. A total of 211 patients underwent SLN mapping. Of these, 165 patients (78%) were female and 46 (22%) were male. Also, 75 (36%) were ≤45 years of age, and 136 (64%) were older than 45. Tumors were ≤2.0 cm (T1) in 142 patients (67%), 2-4 cm (T2) in 35 patients (17%), &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;4 cm with minimal invasion (T3) in 32 patients (15%), and locally invasive (T4) in 2 patients (1%). At least 1 blue node was found in 192 patients (91%). Also, 47 patients had a positive SLN on frozen section, with an additional 24 node-positive patients on permanent section, for a total of 71 (37%). There were 43 patients (91%) who underwent central neck dissection; 26 (60%) had additional metastases. Sentinel lymphadenectomy for papillary thyroid carcinoma is feasible, safe, and can identify patients who may benefit from central neck dissection.

Calcified Tissue International, 2008

Paget's disease of bone (PDB) is a localized bone disease characterized by excessive bone resorpt... more Paget's disease of bone (PDB) is a localized bone disease characterized by excessive bone resorption due to overactive osteoclasts. Seven genetic loci (PDB1-PDB7) have been reported for late-onset PDB. PDB3 is the only locus where a gene, sequestosome 1 (SQSTM1), has been identified. Mutations in SQSTM1 have been associated with both sporadic and hereditary PDB in different populations. However, the SQSTM1 mutation frequency in PDB patients from a more heterogeneous population has never been reported. To investigate this, we determined the frequency of mutations in patients from the United States. Blood was collected from sporadic and hereditary PDB patients in the United States. DNA was isolated from whole blood or from serum. The SQSTM1 sequence was determined for exons and intron/exon junctions from whole blood and serum. A total of 112 (39 hereditary, 73 sporadic) samples were collected. Eight mutations were found in hereditary PDB patients, for a mutation frequency of 20.5% (95% confidence interval [CI] 10.8-35.5%) and did not differ significantly from mutation rates observed in studies in Canada, Great Britain, and The Netherlands. No mutations were found in sporadic patients, for a frequency of 0% (95% CI 0.0-5.0%), which was statistically significantly lower than the mutation rates previously observed in populations from Australia (P = 0.009), Canada (P = 0.008), Great Britain (P = 0.02), and France (P = 0.04) but not compared to rates from Belgium, The Netherlands, and Italy. Four out of five families with the P392L mutation carried it on the H2 haplotype. Mutations in SQSTM1 seem to contribute to the pathogenesis of PDB

Clinical Chemistry, 1997

Previous studies have shown that immunoassay of urinary NTx (cross-linked N-telopeptides of type ... more Previous studies have shown that immunoassay of urinary NTx (cross-linked N-telopeptides of type I collagen) provides a responsive index of human bone resorption. Here we report by a sensitive immunoassay that NTx is present in serum and is suppressed appropriately in patients with Paget disease of bone by bisphosphonate antiresorptive therapy. The monoclonal antibody (1H11) developed against urinary NTx was applied in a sensitive chemiluminescence format. Results for human serum and urine showed parallel inhibition curves. The NTx concentrations in paired serum and urine samples from individual patients correlated well when urinary concentrations were normalized to creatinine concentrations (in premenopausal and postmenopausal women and Paget disease patients, r = 0.90, n = 60). The percentage of NTx suppression from baseline values for Paget disease patients on bisphosphonate therapy was similar for serum and urine. Blood samples drawn from bone marrow at the site of Pagetic lesio...

Clinical Chemistry, 1994

This two-site IRMA includes specific monoclonal antibodies for measuring skeletal alkaline phosph... more This two-site IRMA includes specific monoclonal antibodies for measuring skeletal alkaline phosphatase (B-ALP) in human serum. Assay calibration is based on mass units (micrograms per liter) and was established with purified B-ALP from a human osteosarcoma cell line, SAOS-2. Precision studies demonstrated intra- and interassay CVs of 3-5% and 5-7%, respectively. Relative reactivity studies showed that the assay has a sevenfold preference for detecting B-ALP compared with the liver isoenzyme in serum. The normal reference interval for 478 healthy adults was 5-22 micrograms/L. Method comparison studies showed good correlation between this B-ALP assay (y) and commercially available electrophoretic methods (x) (y = 0.3540x + 20.5, R2 = 0.929) in a pagetic population. Temporal profiles for total ALP, this IRMA B-ALP assay, and B-ALP by electrophoresis in three pagetic patients were parallel. We conclude that this assay demonstrates good analytical performance and would be useful for the ...

Blood, 1989

Fifteen patients with lymphoma and hypercalcemia (greater than or equal to 11.0 mg/dL) were ident... more Fifteen patients with lymphoma and hypercalcemia (greater than or equal to 11.0 mg/dL) were identified by screening the serum chemistry profile obtained from patients upon admission to the Los Angeles County/USC Medical Center. Seven of the 15 (47%) possessed a frankly elevated serum concentration of 1,25-dihydroxyvitamin D [1,25-(OH)2-D]. An additional patient with severe hypercalcemia (16.2 mg/dL) had a serum 1,25-(OH)2-D concentration in the midnormal range, not a suppressed value. To examine the potential existence of hypercalciuria in absence of overt hypercalcemia, prospective screening of 23 normocalcemic patients with lymphoma was undertaken. Four of the 23 patients (17%) had increased fractional urinary calcium excretion rates (0.35 +/- 0.3 mg calcium/100 mL glomerular filtrate [GF], mean +/- SE; normal, less than 0.16 mg/100 mL GF); two of the hypercalciuric patients had a frankly elevated serum 1,25-(OH)2-D concentration. Of the 19 hypercalcemic/hypercalciuric lymphoma pa...

Cleveland Clinic Journal of Medicine, 2008

Biochemical markers of bone turnover provide clinically useful evidence of the normal and patholo... more Biochemical markers of bone turnover provide clinically useful evidence of the normal and pathologic processes that reflect bone cell activity in the skeleton. Understanding the behavior of markers of bone formation and bone resorption should aid in managing patients with a variety of skeletal disorders.

Cancer research, Jan 15, 2001

The presence of skeletal metastases in patients suffering from cancer leads to a variety of clini... more The presence of skeletal metastases in patients suffering from cancer leads to a variety of clinical complications. Bisphosphonates are a class of drugs with a potent bone resorption inhibition activity that have found increasing utility in treating and managing patients with metastatic bone disease. Several clinical trials have demonstrated that bisphosphonates have clinical value in the treatment and management of skeletal metastases derived from advanced prostate cancer. Currently, the mechanism(s) through which bisphosphonates exert their activity is only beginning to be understood. We have studied the effects of bisphosphonate treatment on the growth of prostate cancer cell lines in vitro. Treatment of PC3, DU145, and LNCaP cells with pamidronate or zoledronate significantly reduced the growth of all three cell lines. Using flow cytometry, pamidronate treatment (100 microM) was shown to induce significant amounts of cell death in all three cell lines studied. In contrast, treat...

The American journal of pathology, 1997

A question and answer pamphlet concerning fibrous dysplasia is available for patients and physici... more A question and answer pamphlet concerning fibrous dysplasia is available for patients and physicians from The Paget Foundation, 120 Wall Street, Suite 1602, New York, NY 10005.

Proceedings of the National Academy of Sciences, 1981

Respiratory syncytial virus antisera have been found to produce a positive immunohistologic respo... more Respiratory syncytial virus antisera have been found to produce a positive immunohistologic response in osteoclasts in bone sections or in cells cultured from Paget disease lesions in 12 out of 12 patients tested. These experiments were carefully controlled by several means. Use of experimentally infected cells served as positive controls. Adsorption of antisera on human bone powder and KB cells did not remove the specific immunologic stain, but adsorption of the antisera by the virus did. Negative results were also obtained in osteoclasts of patients with primary or secondary hyperparathyroidism. In addition, negative results in specimens of Paget disease were found with antisera to measles; parainfluenza 1, 2, and 3; influenza A, B and C; rubella; and herpes simplex. These results are consistent with the hypothesis that the nuclear and cytoplasmic inclusions in the osteoclasts of Paget disease are a result of viral activity.

Kidney International, 1986

The effects of metabolic acidosis on hone formation and bone resorption in the rat. Metabolic aci... more The effects of metabolic acidosis on hone formation and bone resorption in the rat. Metabolic acidosis (MA) has been implicated in the pathogenesis of both osteomalacia and osteopenia. Alterations in the secretion of parathyroid hormone and in the metabolism of vitamin D may contribute to such skeletal changes. To minimize the influence of these factors, quantitative bone histology and measurements of bone formation using double tetracycline labeling were done in thyroparathyroidectomized (TPTX) rats with MA induced by ammonium chloride (TPTX-A), and in both non-acidotie TPTX (TPTX-C) and intact (C) controls. To evaluate the response of both cortical and trabecular bone to MA, histologic studies were done at three separate sites in the tibia, cortical bone from the mid-shaft, and trabeeular bone from the epiphysis and from the metaphysis. Plasma pH was lower in TPTX-A, 7.24 0.10, than in either TPTX-C, 7.39 0.03, or C, 7.43 0.04, P <0.01, and urinary hydroxyproline excretion increased from 89.8 8.7 in TPTX-C to 150.2 25.9 pg/mg/ereatinine in TPTX-A, P < 0.01. Resorption surface at the epiphysis increased from 1.8 0.6% in TPTX-C to 4.0 1.6% in TPTX-A, P < 0.05, values not different from those in C, 3.1 1.1%. Resorption surface was unchanged at other skeletal sites, but total bone volume at the metaphysis fell from 15.5 5.6% in TPTX-C to 9.0 4.3% in TPTX-A, P < 0.05. Bone formation was reduced at each skeletal site in TPTX-A vs. TPTX-C, P < 0.05 for all values, but histologic evidence of osteomalacia was not observed. Thus, bone resorption is enhanced and bone formation is impaired in MA. Both changes may contribute to osteopenia, but osteomalaeia is not a direct consequence of MA. Metabolic acidosis is associated with an increase in the urinary excretion of calcium and with negative skeletal calcium balance both in man and in experimental animals [1-3]. These changes may result in the development of osteopenia [41. It has been suggested that calcium is mobilized from the skeleton in metabolic acidosis either through the dissolution of bone mineral directly [5-7] or by the stimulation of cell-mediated bone resorption [8, 9]. Several investigators have reported evidence of enhanced cell-mediated bone resorption in morphologic studies of bone in acidptic rats [4, 91. Osteopenia can also develop due to disturbances in the accretion of mineral into bone [101. Such changes may occur as a consequence of either reductions in the rate of bone formation or defects in the mineraiization of osteoid [10]. The latter disturbance leads to

Journal of Clinical Investigation, 2007

Paget disease is the most exaggerated example of abnormal bone remodeling, with the primary cellu... more Paget disease is the most exaggerated example of abnormal bone remodeling, with the primary cellular abnormality in the osteoclast. Mutations in the p62 (sequestosome 1) gene occur in one-third of patients with familial Paget disease and in a minority of patients with sporadic Paget disease, with the P392L amino acid substitution being the most commonly observed mutation. However, it is unknown how p62 P392L mutation contributes to the development of this disease. To determine the effects of p62 P392L expression on osteoclasts in vitro and in vivo, we introduced either the p62 P392L or WT p62 gene into normal osteoclast precursors and targeted p62 P392L expression to the osteoclast lineage in transgenic mice. p62 P392L-transduced osteoclast precursors were hyperresponsive to receptor activator of NF-κB ligand (RANKL) and TNF-α and showed increased NF-κB signaling but did not demonstrate increased 1,25-(OH) 2 D 3 responsivity, TAF II-17 expression, or nuclear number per osteoclast. Mice expressing p62 P392L developed increased osteoclast numbers and progressive bone loss, but osteoblast numbers were not coordinately increased, as is seen in Paget disease. These results indicate that p62 P392L expression on osteoclasts is not sufficient to induce the full pagetic phenotype but suggest that p62 mutations cause a predisposition to the development of Paget disease by increasing the sensitivity of osteoclast precursors to osteoclastogenic cytokines.

Journal of Clinical Investigation, 2000

Paget's disease is a highly localized process in which large numbers of abnormal osteoclasts (OCL... more Paget's disease is a highly localized process in which large numbers of abnormal osteoclasts (OCLs) induce increased bone resorption (1, 2). The primary cellular abnormality in Paget's disease resides in the OCLs. The OCLs are increased in number and size, have increased nuclei per multinucleated cell, contain paramyxoviral-like nuclear inclusions (3), and are hyperresponsive to 1,25-dihydroxyvitamin D3, or 1,25-(OH) 2 D 3 (4). They also express high levels of osteotropic factors that can regulate OCL activity, including IL-6 (5), IL-6 receptor, and NF-κB (6). In addition, A.P. Mee (7) has reported that the BCL2 gene is overexpressed in OCLs from patients with Paget's disease, suggesting that the OCL life span may be prolonged in pagetic lesions. However, the basis for the increased OCL formation and the highly localized nature of Paget's disease is still unknown. Recently, RANK ligand (RANKL), a newly described member of the TNF family, has been identified as a critical osteoclastogenic factor (8-10). RANKL is expressed on marrow stromal cells and osteoblasts, and appears to mediate the effects of most osteoclastogenic factors. In vivo studies have shown that mice lacking RANKL developed severe osteopetrosis (11). Factors such as 1,25-(OH) 2 D 3 , IL-1, IL-11, and prostaglandin E 2 appear to induce OCL formation indirectly by upregulating RANKL expression on marrow stromal cells (12). These data suggest that RANKL may be the common mediator for the effects of most osteotropic factors on OCL formation. Therefore, we have examined the relative levels of RANKL expression by marrow stromal cells from normal bones and affected bones from patients with Paget's disease, and the RANKL responsivity of OCL precursors from normal bones and affected bones from Paget's patients. Methods These studies were approved by the Institutional Review Board at the University of Texas Health Science Center at San Antonio and the Auckland Ethics Committee. OCL culture. Nonadherent human bone marrow cells were collected from eight normal volunteers. Samples were also taken from involved bones of six patients with Paget's disease, and both involved and uninvolved bone from two other Paget's patients. Cells were pre

Journal of Clinical Investigation, 1977

This is publication no. 724 of the Robert W. Lovett Memorial Group for the Study of Diseases Caus... more This is publication no. 724 of the Robert W. Lovett Memorial Group for the Study of Diseases Causing Deformities.

Journal of Clinical Investigation, 1991

Parathyroid hormone-related protein (PTHrP), which is responsible for producing hypercalcemia in ... more Parathyroid hormone-related protein (PTHrP), which is responsible for producing hypercalcemia in patients with humoral hypercalcemia of malignancy, has recently been identified in several normal tissues. Because PTHrP, like parathyroid hormone (PTH), is known to exhibit vasodilatory properties, we investigated the expression and regulation of PTHrP mRNA in cultured rat aortic smooth muscle cells (SMC). We report here that PTHrP mRNA is expressed in SMC and is markedly induced by serum in a timeand concentration-dependent fashion. Addition of 10% fetal calf serum to serum-deprived, confluent cells, resulted in a marked induction of PTHrP mRNA by 2 h with a peak at 4-6 h. PTHrP was detected in SMC by immunocytochemistry and radioimmunoassay of conditioned medium, and was shown to be up-regulated within 24 h after the addition of serum. The serum induction of PTHrP mRNA was blocked by actinomycin D and by cycloheximide indicating the need for protein synthesis to evoke the serum effect on PTHrP gene transcription. In addition, treatment with dexamethasone, which has been previously shown to reduce the constitutive expression of PTHrP in human cancer cells, also blunted the serum induction of PTHrP mRNA in SMC. Treatment of quiescent cells with the serum mitogens platelet-derived growth factor or insulin-like growth factor-I had no effect on PTHrP, whereas the vasoactive peptides endothelin, norepinephrine and thrombin stimulated PTHrP expression. Exogenous addition of recombinant PTHrP41-141) had no significant effect on SMC DNA synthesis as measured by [3HJthymidine incorporation. In summary, the abundance of PTHrP mRNA and the characteristics of its regulation in SMC suggest a major role for PTHrP as a local modulator in vascular smooth muscle.