Effects of estradiol and tamoxifen on creatine kinase in rodent mammary carcinomas.

The effects of altered estrogenic environments on creatine kinase (CK) and adenylate kinase (AK) were studied in two rodent mammary tumor systems, R3230AC and primary 7,12-dimethylbenz(a)anthracene (DMBA)-induced carcinomas, to determine whether response of these enzymes could be related to their hormone dependence. The hormonal perturbations studied were ovariectomy and administration of various doses of estradiol valerate or the antiestrogen tamoxifen. Total CK activity and AK activity were assessed by a spectrophotometric assay followed by electrophoretic separation of the CK isozymes to determine their relative activities. In the ovarian-independent R3230AC tumors, estrogen treatment produced a dose-related decrease in CK activity, whereas CK was not responsive in ovarian-dependent DMBA-induced tumors. Adenylate kinase activity remained unchanged regardless of the hormonal perturbation. Glucose-6-phosphate dehydrogenase and lactate dehydrogenase, which were studied for comparative purposes, were both estrogen responsive. While both estrogenic and antiestrogenic effects on enzyme activities were observed in the DMBA-induced tumors, the effect of tamoxifen in the R3230AC tumors was generally estrogenic. We conclude that the effect of estrogen on CK-BB in DMBA-induced tumors is not sufficient to be used as a biochemical marker of hormone dependence.

Fields of aberrant CpG island hypermethylation in Barrett's esophagus and associated adenocarcinoma.

Esophageal adenocarcinoma (EAC) is thought to develop through a multistage process in which Barrett's metaplasia progresses through low- and high-grade dysplasia to invasive cancer. Transcriptional silencing of tumor suppressor genes by promoter CpG island hypermethylation has been observed in many types of human cancer. Analysis of CpG island hypermethylation in EAC has thus far been limited to the CDKN2A (p16) gene. In this study, we extend the methylation analysis of EAC to include three other genes, APC, CDH1 (E-cadherin), and ESR1 (ER, estrogen receptor alpha), in addition to CDKN2A. Molecular analysis can provide insight into the complex relationships between tissues with different histologies in Barrett's esophagus and associated adenocarcinoma. Therefore, we have mapped the spatial distribution of methylation patterns in six esophagectomy cases in detail. Hypermethylation of the four CpG islands was analyzed by the MethyLight technique in 107 biopsies derived from these six patients for a total of 428 methylation analyses. Our results show that normal esophageal squamous epithelium is unmethylated at all four CpG islands. CDH1 is unmethylated in most other tissue types as well. Hypermethylation of ESR1 is seen at high frequency in inflammatory reflux esophagitis and at all subsequent stages, whereas APC and CDKN2A hypermethylation is found in Barrett's metaplasia, dysplasia, and EAC. When it occurs, hypermethylation of APC, CDKN2A, and ESR1 is usually found in a large contiguous field, suggesting either a concerted methylation change associated with metaplasia or a clonal expansion of cells with abnormal hypermethylation.

Epigenetic patterns in the progression of esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) arises after normal squamous mucosa undergoes metaplasia to specialized columnar epithelium (intestinal metaplasia or Barrett's esophagus), which can then ultimately progress to dysplasia and subsequent malignancy. Epigenetic studies of this model have thus far been limited to the DNA methylation analysis of a few genes. In this study, we analyzed a panel of 20 genes using a quantitative, high-throughput methylation assay, METHYLIGHT: We used this broader approach to gain insight into concordant methylation behavior between genes and to generate epigenomic fingerprints for the different histological stages of EAC. Our study included a total of 104 tissue specimens from 51 patients with different stages of Barrett's esophagus and/or associated adenocarcinoma. We screened 84 of these samples with the full panel of 20 genes and found distinct classes of methylation patterns in the different types of tissue. The most informative genes were those with an intermediate frequency of significant hypermethylation [ranging from 15% (CDKN2A) to 60% (MGMT) of the samples]. This group could be further subdivided into three classes, according to the absence (CDKN2A, ESR1, and MYOD1) or presence (CALCA, MGMT, and TIMP3) of methylation in normal esophageal mucosa and stomach, or the infrequent methylation of normal esophageal mucosa accompanied by methylation in all normal stomach samples (APC). The other genes were less informative, because the frequency of hypermethylation was below 5% (ARF, CDH1, CDKN2B, GSTP1, MLH1, PTGS2, and THBS1), completely absent (CTNNB1, RB1, TGFBR2, and TYMS1), or ubiquitous (HIC1 and MTHFR), regardless of tissue type. Each class undergoes unique epigenetic changes at different steps of disease progression of EAC, suggesting a step-wise loss of multiple protective barriers against CpG island hypermethylation. The aberrant hypermethylation occurs at many different loci in the same tissues, suggestive of an overall deregulation of methylation control in EAC tumorigenesis. However, we did not find evidence for a distinct group of tumors with a CpG island methylator phenotype. Finally, we found that normal and metaplastic tissues from patients with evidence of associated dysplasia or cancer had a significantly higher incidence of hypermethylation than similar tissues from patients with no further progression of their disease. The fact that the samples from these two groups of patients were histologically indistinguishable, yet molecularly distinct, suggests that the occurrence of such hypermethylation may provide a clinical tool to identify patients with premalignant Barrett's who are at risk for further progression.

The management of ductal carcinoma in situ of the breast.

Ductal carcinoma in situ (DCIS) of the breast is a heterogeneous group of lesions with diverse malignant potential. It is the most rapidly growing subgroup within the breast cancer family with more than 42 000 new cases diagnosed in the United States during 2000. Most new cases are nonpalpable and are discovered mammographically. Treatment is controversial and ranges from excision only, to excision with radiation therapy, to mastectomy. Prospective randomized trials reveal an approximate 50% reduction in local recurrence rate overall with the addition of radiation therapy to excisional surgery, but the published prospective data do not allow the selection of subgroups in whom the benefit from radiation therapy is so small that its risks outweigh its benefits. Nonrandomized single facility series suggest that age, family history, nuclear grade, comedo-type necrosis, tumor size and margin width are all important factors in predicting local recurrence and that one or more of these factors could be used to select subgroups of patients who do not benefit sufficiently from radiation therapy to merit its use. When all patients with ductal carcinoma in situ are considered, the overall mortality from breast cancer is extremely low, only about 1-2%. When conservative treatment fails, approximately 50% of all local recurrences are invasive breast cancer. In spite of this, the mortality rate following invasive local recurrence is relatively low, about 12% with eight years of actuarial follow-up. Genetic changes routinely precede morphological evidence of malignant transformation. Lessons learned from ongoing basic science research will help us to identify those DCIS lesions that are unlikely to progress and to prevent progression in the rest.

The role of surgery in the treatment of nonregionally recurrent melanoma.

BACKGROUND: Between 1971 and 1989, 144 patients underwent 183 surgical procedures for resection of nonregional metastatic melanoma. METHODS: Thirty-six patients with subcutaneous or nodal metastases, 46 patients with pulmonary metastases, 17 patients with brain metastases, 19 with gastrointestinal metastases, and 26 patients with miscellaneous sites of metastatic involvement underwent resection. RESULTS: The overall 5- and 10-year actuarial survival rates were 20% and 14%, respectively. A single postoperative death occurred in a patient operated on for palliative treatment of gastrointestinal metastases. CONCLUSIONS: Cox regression analysis suggests that patients with a solitary metastasis confined to the subcutaneous, nonregional lymph nodes or lung are most likely to benefit from aggressive surgical intervention. Surgical intervention represents a potentially important modality in the management of selected patients with nonregional metastatic melanoma.

Recurrent secondary hyperparathyroidism. An argument for total parathyroidectomy.

OBJECTIVE: To define better the incidence and causes of recurrent secondary hyperparathyroidism. DESIGN: Review of total parathyroidectomy with autotransplantation in uremic patients and literature review. SETTING: Two teaching hospitals. PATIENTS: Nine patients treated for secondary hyperparathyroidism between 1982 and 1993 by a single surgeon. INTERVENTIONS: Total parathyroidectomy with autotransplantation into the sternocleidomastoid muscle. Recurrence was treated, if necessary, with a graft reduction procedure. MAIN OUTCOME MEASURES: The symptomatic and biochemical response to initial therapy, morbidity, and mortality, as well as the development of recurrent hypercalcemia. RESULTS: All patients were normocalcemic after their initial surgery. One patient died postoperatively (mortality, 11%). Three (38%) of the remaining patients developed recurrent hypercalcemia, 2 (25%) requiring reoperation. Of the 2 patients who underwent surgery for recurrence, 1 had an adenoma in the implant and the other had graft hyperplasia. CONCLUSIONS: Recurrence rates after total parathyroidectomy with autotransplantation are substantial and, given the pathophysiology of secondary hyperparathyroidism, unavoidable in patients with uncorrected renal failure. An argument is made for performing total parathyroidectomy alone in patients with secondary hyperparathyroidism who will not undergo renal transplantation in the near future.

Surgical treatment and chemotherapy for pulmonary metastases from osteosarcoma.

Between 1971 and 1991, 247 patients with stage I osteosarcoma were treated at UCLA. Patients were treated in four sequential groups, with group 1 receiving surgery alone, and groups 2 through 4 receiving various adjuvant chemotherapeutic regimens. The incidence of lung metastases in these patients decreased from 92% (group 1) to 31% (group 4), while the proportion of patients undergoing pulmonary resection increased (17% vs 82%). Overall 5-year survival rate among patients with pulmonary metastases increased from 0 in group 1 to 41% (actuarial) in group 4. No clinical factor correlated significantly with outcome using univariate analysis, although there was a trend toward prolonged survival in those with longer disease-free intervals. Adjuvant chemotherapy and resection of pulmonary metastases have transformed a uniformly fatal condition into one with a reasonable expectation of long-term survival.

Prospective evaluation of the use of antigen-specific immune complexes in predicting the development of recurrent melanoma.

We previously demonstrated that the antigen-specific immune complexes captured by the monoclonal antibody MAb JSI in a sandwich enzyme-linked immunosorbent assay were associated with recurrent melanoma. To determine the potential use of antigen-specific immune complex analysis in predicting the development of recurrent melanoma, we prospectively analyzed serum obtained from patients with melanoma following surgical treatment. Two hundred fifty-three patients have been followed up for a median of 25 months (range, 17 to 29 months). Seventy-seven patients (30%) have developed recurrent melanoma. Antigen-specific immune complexes correlated with the stage of disease at time of entry into the study. The absence of antigen-specific immune complexes in postoperative serum samples is predictive of a disease-free status. Long-term follow-up will define the false-positive rate of antigen-specific immune complex analysis. Continued refinement of this approach should lead to clinically useful methodology to monitor human melanoma.

Telomerase reverse transcriptase expression is increased early in the Barrett's metaplasia, dysplasia, adenocarcinoma sequence.

Barrett's esophagus is a multistage polyclonal disease that is associated with the development of adenocarcinoma of the esophagus and esophagogastric junction. Telomerase activation is associated with cellular immortality and carcinogenesis, and increased expression of the telomerase reverse transcriptase catalytic subunit (hTERT) has been used for the early detection of malignant diseases. To identify biomarkers associated with each stage of the Barrett's process, relative mRNA expression levels of hTERT were measured using a quantitative reverse transcription-polymerase chain reaction method (ABI 7700 Sequence Detector (TaqMan system) in Barrett's intestinal metaplasia (n = 14), Barrett's dysplasia (n = 10), Barrett's adenocarcinoma (n = 14), and matching normal squamous esophagus tissues (n = 32). hTERT expression was significantly increased at all stages of Barrett's esophagus, including the intestinal metaplasia stage, compared to normal tissues from patients without cancer (intestinal metaplasia vs. normal esophagus, P <0.0001; dysplasia, P = 0.001; adenocarcinoma, P = 0.007; all Mann-Whitney U test ). hTERT expression levels were significantly higher in adenocarcinoma tissues than in intestinal metaplasia tissues (P = 0.003), and were higher in dysplasia compared with intestinal metaplasia tissues (P = 0.056). hTERT levels were also significantly higher in histologically normal squamous esophagus tissues from cancer patients than in normal esophagus tissues from patients with no cancer (P = 0.013). Very high expression levels ([hTERT x 100: beta-actin] >20) were found only in patients with cancer. These findings suggest that telomerase activation is an important early event in the development of Barrett's esophagus and esophageal adenocarcinoma, that very high telomerase levels may be a clinically useful biomarker for the detection of occult adenocarcinoma, and that a widespread cancer "field" effect is present in the esophagus of patients with Barrett's cancer.

High negative appendectomy rates are no longer acceptable.

BACKGROUND: A 10% to 20% negative appendectomy rate has been accepted in order to minimize the incidence of perforated appendicitis with its increased morbidity. We reviewed our experience with appendicitis in order to determine the incidence of negative appendectomies and perforation, and the role of delay in diagnosis or treatment. METHODS: We reviewed 659 appendectomies performed over a 12-month period. Incidental and pediatric appendectomies were excluded. RESULTS: Seventy-five percent of patients were male and 25% female. Nine percent had negative appendectomies and 28% had perforated appendicitis. Perforated appendicitis resulted in increased morbidity and length of stay. Delay in presentation greater than 12 hours after the onset of symptoms significantly increased the perforation rate. In-hospital delay did not affect perforation rate. CONCLUSIONS: We have achieved a negative appendectomy rate lower than that in other reported series, while maintaining an acceptable perforation rate. In the majority of patients, perforated appendicitis is a result of late presentation.

Preoperative 5-fluorouracil and radiation therapy for locally advanced breast cancer.

BACKGROUND: Fifteen percent of breast cancer patients present with large tumors involving skin or chest wall. Often, surgery with primary wound closure is impossible. We used neoadjuvant chemoradiation in locally advanced breast cancer patients, in hopes of increasing resectability. METHODS: Eligible patients had locally advanced breast cancer deemed unresectable with primary wound closure. Patients received 8 weeks of infusional 5-fluorouracil (5-FU) 200 mg/m2 per day and radiation therapy to 50 Gy. Patients rendered resectable underwent modified radical mastectomy (MRM) followed up by chemotherapy. RESULTS: Of 30 evaluable patients, 73% had an objective clinical response. All were able to undergo MRM with primary wound closure; 63% had residual disease, 20% had minimal microscopic disease, and 17% had complete pathologic response. Treatment-related toxicity was minimal. Surgical morbidity was not increased. CONCLUSIONS: Infusional 5-FU with concomitant radiotherapy is well tolerated and effective at producing shrinkage in the majority of patients, converting inoperable breast cancer to easily resectable disease.

Recurrent Zenker's diverticulum: treatment with crycopharyngeal myotomy.

Recurrence following treatment of Zenker's diverticulum (ZD) occurs in up to 16 per cent of patients. We have reviewed our experience with cricopharyngeal myotomy (CM) to determine its safety and efficacy in the treatment of recurrent ZD. Eight patients were treated, five with early recurrence (symptoms persisting or recurring within 6 months of their initial surgery) and three with late recurrence. Most patients with early recurrence did not have an adequate CM as part of their initial therapy, suggesting that adequate myotomy is important for early relief of dysphagia. Seven patients underwent CM alone, and one patient underwent CM with diverticulectomy. All patients experienced immediate relief of their dysphagia, with good to excellent results persisting at last follow-up (mean follow-up 53 months). Complications were seen only in the patient who underwent combined myotomy with diverticulectomy. We have found CM alone to be quite safe and effective in the treatment of recurrent ZD.

Recurrent secondary hyperparathyroidism after autotransplantation into the sternocleidomastoid muscle: report of a case with fine needle aspiration findings.

BACKGROUND: Recurrent hyperparathyroidism may occur following parathyroid autotransplantation due to autogenous function of the muscle-engrafted tissue. Parathyroid lesions are uncommonly diagnosed on cytology. CASE: A 31-year-old female with chronic renal failure presented with an elevated parathyroid hormone level and a neck mass in the left sternocleidomastoid muscle, the site of a previous parathyroid autograft. Fine needle aspiration of the mass revealed high cellularity, with perivascularly arranged, three-dimensional, branching clusters; individual cells; and naked nuclei exhibiting anisonucleosis. A diagnosis of parathyroid graft hyperplasia was made by fine needle aspiration and subsequently by histopathologic examination. CONCLUSION: Fine needle aspiration is an effective tool for confirming the presence of parathyroid autograft hyperplasia, thus allowing the correct surgical approach.

Changes in the synthesis and metabolism of prostaglandins by human fetal membranes and decidua at labor.

The production of prostaglandins by dispersed cells from human amnion, chorion, and decidua was examined at term before the onset of labor and at spontaneous vaginal delivery. In order to obtain detailed information about relative prostaglandin production rates, the time course of prostaglandin output was examined by incubating the cells for up to 4 hours and measuring the cumulative output of prostaglandin E2, prostaglandin F2 alpha and 13,14-dihydro-15-keto-prostaglandin F2 alpha in the incubation media. The output of all three prostaglandins was low in tissues obtained before the onset of labor. At labor there was an increased production of prostaglandins E2 and F2 alpha in amnion and a small increase in the output of prostaglandin E2, prostaglandin F2 alpha, and 13,14-dihydro-15-keto-prostaglandin F2 alpha in decidua. In contrast, chorionic cells obtained at spontaneous vaginal delivery showed high levels of 13,14-dihydro-15-keto-prostaglandin F2 alpha in the media with no net production of prostaglandin E2 or F2 alpha. These data suggest a high rate of specific in vitro prostaglandin synthesis in amnion and decidua at labor, accompanied by a high rate of prostaglandin metabolism in chorion.

Influence of desalivation on acid secretory output and gastric mucosal integrity in the rat.

Exogenous administration of substances extracted from rodent salivary glands may increase the resistance of gastric mucosal barrier to the disruptive actions of ulcerogenic agents and/or reduce acid secretory output. In the present study the influence of desalivation in the rat on acid secretory function and mucosal integrity has been investigated. Experiments were performed on rats 4 wk after removal of the major salivary glands and ligation of the salivary ducts. Intravenous infusion of pentagastrin (0.1--0.4 microgram/kg/min) resulted in an increase in acid output in desalivated rats although the responses was less than that observed in sham-operated controls. Intraluminal instillation of a bile salt solution (5 mM in 150 mM HCl) in control animals resulted in a significant loss of H+ and the luminal appearance of Na+ and K+. The net fluxes of Na+ and K+ were exacerbated in the desalivated group. Furthermore, the mean area of bile salt-induced ulceration was significantly greater in desalivated rats than in the control group. In the absence of bile salt treatment there was no difference in either the transmural ionic fluxes or area of ulceration between the two groups. These results suggests that removal of the major salivary glands in rats decreases the resistance of the gastric mucosa to bile salt-induced damage.

Effect of sialoadenectomy and salivary gland extracts on gastrointestinal mucosal growth and gastrin levels in the rat.

We have examined gastrointestinal mucosal growth 30 days after surgical removal of the submandibular-sublingual salivary glands and ligation of the parotid ducts of rats. The rate of [3H]thymidine uptake in vitro as an estimation of DNA synthesis and the content of DNA and RNA were examined in the oxyntic, duodenal and proximal colonic mucosa. DNA synthesis, DNA and RNA content of oxyntic mucosa were reduced in sialoadenectomized rats when compared to sham-sialoadenectomized control animals. There was no change in the degree of [3H]thymidine incorporation or DNA content of the duodenal or colonic mucosa. Intraperitoneal injection of an aqueous extract of the submandibular-sublingual salivary glands of rats (4.0 mg tissue protein in 0.1 M-sodium phosphate buffer administered twice a day for 15 days) increased the rate of DNA synthesis and the total mucosal DNA and RNA content in the oxyntic mucosa. Injections of extracts of spleen or muscle did not produce consistent results. Administration of epidermal growth factor (10 micrograms/kg) or pentagastrin (250 micrograms/kg) resulted in an increase of the level of DNA synthesis observed in the oxyntic mucosa of sialoadenectomized rats. Plasma and antral tissue levels of the trophic hormone, gastrin, were not significantly decreased in sialoadenectomized rats treated with 0.1 M-sodium phosphate-buffered saline. However, treatment with the salivary tissue extract did result in significant reductions in both plasma and tissue levels of gastrin. We conclude that elimination of the major salivary glands in the rat results in a decrease in [3H]thymidine uptake and DNA content of the gastric oxyntic mucosa. These effects are not mediated via a reduction in endogenous levels of the trophic hormone, gastrin. Administration of an aqueous extract of salivary tissue exerted a small but significant trophic influence on the oxyntic mucosa of the rat.

Predicting axillary nodal positivity in 2282 patients with breast carcinoma.

Axillary lymph node status continues to be the single most important prognostic variable for breast cancer survival despite significant progress in the molecular and genetic characterization of breast malignancies. All patients with invasive breast cancer who underwent axillary lymph node dissection as part of their treatment were evaluated by 11 clinical and pathologic factors, including the primary lesion's T category (TNM staging system), whether the lesion was clinically palpable, the presence of lymphatic or vascular invasion, nuclear grade, estrogen and progesterone receptors, S-phase, age, HER2/neu overexpression, histology (infiltrating lobular or ductal), and ploidy. A total of 2282 axillary dissections were performed: 391 in patients with ductal carcinoma in situ (DCIS) [3 of which (0.8%) contained metastases] and 1891 in patients with invasive breast cancer [680 of which (36%) contained metastases]. Multivariate analysis of patients with invasive cancer identified four factors as independent predictors of axillary lymph node metastases: lymph/vascular invasion, tumor size, nuclear grade, tumor palpability. Among a group of 189 patients with nonpalpable, non-high-grade invasive lesions 15 mm or smaller without lymph/vascular invasion, only 6 (3%) had metastases to lymph nodes. If any three of the favorable factors were present, lymph node positivity was 6% or less. Clinical and pathologic feature of the primary lesions can be used to estimate the risk of axillary lymph node metastases. Such risk assessment can be used for the treatment decision-making process.

Palpable breast cancers are inherently different from nonpalpable breast cancers.

BACKGROUND: We examined the clinicopathologic profile of T1 cancers to determine whether palpable cancers are different from nonpalpable cancers. METHODS: A prospective database was reviewed. Palpable T1 cancers were compared with nonpalpable T1 cancers. Initial significance was determined by chi2 analysis. Factors found to be significant were then reanalyzed. controlling for tumor size by logistic or linear regression, as appropriate. RESULTS: Of 1263 T1 cancers treated between 1981 and 2000, 857 (68%) were palpable and 401 (32%) were nonpalpable. Palpability correlated with pathologic tumor size, mitotic grade, nuclear grade, high S-phase, lymphovascular invasion, nodal positivity, and lack of extensive intraductal component, multifocality, and multicentricity. There was no significant difference in estrogen receptor, progesterone receptor or Her-2/neu status, ploidy, or DNA index. Breast cancer-specific survival was worse for patients with palpable cancers. CONCLUSIONS: Palpable cancers are inherently different from nonpalpable cancers, with a less diffuse growth pattern, higher metastatic potential, higher proliferative activity, more nuclear abnormalities, and a worse prognosis.

Free and protein-associated nitrotyrosine formation following rat liver preservation and transplantation.

Nitrotyrosine in human and animal tissues has been associated with pathologic conditions such as atherosclerosis, renal failure, and acute lung disease. In this study, free and protein-associated nitrotyrosine were determined in plasma and tissue samples using a dual-channel electrochemical detection method. Free nitrotyrosine was quantified in acetonitrile-extracted samples while protein-associated nitrotyrosine was determined in proteinase K-digested samples. In human plasma, total nitrotyrosine increased from 2.3 to 4.3 and 13.2 mumol/mol Tyr following addition of 0, 0.5, and 1 mM ONOO-. To determine if nitrotyrosine was produced during ex vivo hypothermic preservation, rat livers were stored in University of Wisconsin solution (UW) for 0, 6, or 8 h and reperfused for 3 h. Total nitro-tyrosine increased 359 and 908% after 6 and 8 h preservation compared to 0 h. To determine if nitrotyrosine was produced in vivo following hepatic ischemia, a rat preservation-transplantation model was utilized in which livers were flushed with cold UW (0-h group) or transplanted following 6 h hypothermic preservation in UW. Free nitrotyrosine increased from 15.7 +/- 0.3 in the 0-h group to 23.6 +/- 2.5 mumol/mol Tyr, 24 h posttransplant of 6-h preserved livers. Protein-associated nitrotyrosine increased from 9.5 +/- 1.1 in the 0-h group to 27.5 +/- 0.7 mumol/mol Tyr in the 6-h preservation-transplantation group. Protein-associated nitrotyrosine provides an integrative determination of nitration. Detection of free and protein-associated nitrotyrosine in biologic samples may allow insight into the role of .NO-derived oxidants in tissue injury associated with various pathologic conditions.