PDGFR-alpha as a potential therapeutic target in uterine sarcomas.

OBJECTIVE: Uterine sarcomas are a heterogeneous group of tumors with a propensity for metastasis and resistance to conventional therapy. Recent success in the treatment of other solid tumors with the targeted tyrosine kinase inhibitor imatinib mesylate offers new avenues for investigation. The primary target of imatinib is c-kit, but the drug also inhibits PDGFR-alpha and PDGFR-beta. Given the lack of identified molecular targets in endometrial stromal sarcomas, leiomyosarcomas, and carcinosarcomas, the purpose of this study was to determine the protein expression of c-kit, PDGFR-alpha, and PDGFR-beta in these tumors as a preliminary step to determining their susceptibility to directed therapy. A secondary goal was to identify specific gene mutations that might be associated with activation of these proteins in uterine sarcomas. METHODS: Archived tissue from 42 cases of uterine sarcomas was stained for c-kit, PDGFR-alpha, and PDGFR-beta using immunohistochemistry. Laser-capture microdissected samples of uterine carcinosarcomas, or homogeneous areas of leiomyosarcomas or endometrial stromal sarcomas, were subjected to genetic analysis of PDGFR-alpha exons 12 and 18. RESULTS: The majority (38/42, 90%) of uterine sarcomas lacked c-kit expression and 90% (38/42) demonstrated negative or weak staining for PDGFR-beta. In contrast, 70% (30/42) of cases had strong staining for PDGFR-alpha in the tumor but not in normal myometrium or endometrium. Sequencing results revealed no mutations in exons 12 or 18 of PDGFR-alpha. CONCLUSION: c-kit and PDGFR-beta are unlikely to represent primary treatment targets in uterine sarcomas. The strong expression of PDGFR-alpha in uterine sarcoma specimens suggests a role for this receptor in tumor development, although its potential as a therapeutic target requires further investigation.

Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery.

Efficacious bone regeneration could revolutionize the clinical management of bone and musculoskeletal disorders. Although several bone morphogenetic proteins (BMPs) (mostly BMP-2 and BMP-7) have been shown to induce bone formation, it is unclear whether the currently used BMPs represent the most osteogenic ones. Until recently, comprehensive analysis of osteogenic activity of all BMPs has been hampered by the fact that recombinant proteins are either not biologically active or not available for all BMPs. In this study, we used recombinant adenoviruses expressing the 14 types of BMPs (AdBMPs), and demonstrated that, in addition to currently used BMP-2 and BMP-7, BMP-6 and BMP-9 effectively induced orthotopic ossification when either AdBMP-transduced osteoblast progenitors or the viral vectors were injected into the quadriceps of athymic mice. Radiographic and histological evaluation demonstrated that BMP-6 and BMP-9 induced the most robust and mature ossification at multiple time points. BMP-3, a negative regulator of bone formation, was shown to effectively inhibit orthotopic ossification induced by BMP-2, BMP-6, and BMP-7. However, BMP-3 exerted no inhibitory effect on BMP-9-induced bone formation, suggesting that BMP-9 may transduce osteogenic signaling differently. Our findings suggest that BMP-6 and BMP-9 may represent more effective osteogenic factors for bone regeneration.

Outcome and management of pathological stage I endometrial carcinoma patients with involvement of the lower uterine segment.

OBJECTIVE: The objective was to evaluate the clinicopathologic characteristics and outcome of pathologic stage I endometrial carcinoma patients with lower uterine segment (LUS) involvement. METHODS: We retrospectively reviewed the characteristics and outcomes of pathologic stage I endometrial carcinoma patients treated with primary surgery at our institution between 1988 and 1998. The significance of LUS involvement was examined with univariate and multivariate analyses. Median patient follow-up was 37.3 months. RESULTS: Of the 98 cases reviewed, 41 (42%) had LUS involvement. No differences were seen in the clinicopathologic features, extent of surgical staging, or adjuvant therapies between patients with and without LUS involvement. Univariate analysis revealed that grade, lymphovascular invasion (LVI), myometrial invasion (MI), and histology were correlated with recurrence. While the 5-year actuarial disease-free survival was worse in women with LUS involvement (80.3 vs 94.0%) compared to those without, this difference did not reach statistical significance (P = 0.14). Moreover, after controlling for pathologic features in a multivariate model, LUS involvement was not correlated with patient outcome (P = 0.98; hazard rate 0.97; 95% confidence interval 0.24, 4.0). LUS was also not correlated with pelvic recurrence. Of 25 low-risk patients (superficial MI and grade 1-2 disease) with LUS involvement, none recurred in the pelvis following surgery alone. In contrast, pelvic recurrence was common (5/12 or 41.6%) in high-risk patients (deep MI and/or grade 3 tumors) following surgery alone regardless of LUS involvement. CONCLUSION: LUS involvement is common in pathologic stage I endometrial carcinoma but is not correlated with a worse outcome. Moreover, in the absence of adverse pathologic features, LUS involvement is not associated with an increased risk of pelvic recurrence and should not be used as an indication for adjuvant radiation therapy.

Differential expression of mucin genes in mammary and extramammary Paget's disease.

Paget's disease (PD) of the skin is characterized by intraepidermal adenocarcinoma cells, which contain clear cytoplasm and abundant mucin. Nearly all cases of mammary PD (MPD) are associated with underlying ductal carcinoma of the breast, whereas in the majority of cases of extramammary PD (EMPD) no underlying regional malignancy is identified. Mucins are high molecular weight glycoproteins produced by epithelial cells. Different mucin genes are expressed in various types of tissues such as mammary glands, intestinal mucosa, and adnexal structures of the skin. We studied the immunohistochemical expression of apomucin MUC1, MUC2, MUC5AC in MPD, and EMPD. MUC1 is commonly expressed in most cases of PD. MUC5AC is a unique mucin that is exhibited in the majority of cases of EMPD, but not in any MPD. Of the 13 patients with MPD who all had associated breast ductal carcinoma, both Paget cells and underlying ductal carcinoma exhibited the phenotype (MUC1+MUC2-MUC5AC-). This mucin phenotype is also expressed by Toker cells, which have been identified in the epidermis of five of 50 nipples in mastectomies without MPD. Of the three patients with perianal PD who all had associated rectal adenocarcinoma, Paget's cells expressed MUC2 constantly but expressed MUC1 and MUC5AC variably. Seven patients with intraepidermal vulvar PD and two patients with scrotal-penile PD had no identifiable underlying malignancy. Paget cells from all of these nine cases of EMPD expressed a uniform phenotype of mucin (MUC1+MUC2-MUC5AC+). One case of vulvar PD associated with underlying apocrine carcinoma had a phenotype (MUC1+MUC2-MUC5AC-) identical to that of normal apocrine glands. The skin appendage and Bartholin's glands from 20 normal-appearing vulvar skin samples and anal glands from 10 hemorrhoidectomies were also studied. Only Bartholin's gland expressed a mucin phenotype identical to that of intraepidermal EMPD. The results of the present study indicate that 1) MPD may arise from either mammary glands or epidermal Toker cells, 2) intraepidermal EMPD in the anogenital areas may arise from ectopic MUC5AC+ cells originating from Bartholin's or some other unidentified glands, and 3) unique expression of MUC2 in perianal PD indicates its origin from colorectal mucosa. We conclude that the study of mucin gene expression is useful in identifying the histogenesis of PD.

Differential expression of MUC2 and MUC5AC mucin genes in primary ovarian and metastatic colonic carcinoma.

Colonic adenocarcinoma, the most common tumor metastatic to the ovary, may closely mimic primary ovarian adenocarcinoma, especially that of mucinous or endometrioid histology. The differential diagnosis is important for therapeutic considerations. Mucin gene expression is relatively organ-specific and may therefore have use in distinguishing between colonic carcinomas metastatic to the ovary and primary ovarian tumors. In this study, we compared the expression of MUC2 and MUC5AC apomucins in 10 colonic adenocarcinomas metastatic with the ovary, 10 ovarian endometrioid carcinomas (4 primary, 6 metastatic), and 32 primary mucinous ovarian tumors (12 cystadenomas, 10 borderline tumors, and 10 cystadenocarcinomas). Monoclonal antibodies CCP58 and 45M1 were used for immunostains of MUC2 and MUC5AC apomucin, respectively. All but 1 of the 10 metastatic colon adenocarcinomas expressed MUC2, whereas none expressed MUC5AC. None of the 10 endometrioid carcinomas expressed MUC2, and only 2 showed weak immunoreactivity with MUC5AC. All 32 primary mucinous ovarian tumors expressed MUC5AC. The percentages of MUC2-positive immunostaining for cystadenomas, borderline tumors, and cystadenocarcinomas were 0% (0/12), 50% (5/10), and 70% (7/10) respectively. These studies show that MUC2 and MUC5AC are useful markers in the distinction between colonic carcinoma metastatic to the ovary and primary ovarian carcinoma.

Aneurysmal bone cyst of the extremities. Factors related to local recurrence after curettage with a high-speed burr.

BACKGROUND: Aneurysmal bone cyst is a benign, locally destructive lesion of bone. The rates of local recurrence after curettage have varied widely. Therefore, we performed a retrospective study of patients who had had an aneurysmal bone cyst in order to identify the rate of local recurrence and the prognostic factors related to local recurrence after use of contemporary methods of curettage with a high-speed burr. METHODS: We reviewed the cases of forty patients who had been managed by the same surgeon for an aneurysmal bone cyst, as diagnosed on the basis of the latest pathological review, between January 1, 1976, and December 31, 1993. The patients were evaluated with regard to age, gender, the duration and type of symptoms, the presence or absence of pathological fracture, the status of the growth plate, the bone and part of the bone that were involved, the type of operative procedure, the outcome, the radiographic stage, the findings on magnetic resonance imaging and computerized tomography (when it became available) and on bone scintigraphy, and histological parameters. The median duration of follow-up was eighty-seven months (range, fifteen to 267 months). According to the criteria of Enneking, no patient had a stage-1 lesion (one with a surrounding rim of cortical bone), twenty-four had a stage-2 lesion (one with a clearly defined border but no cortical bone), and sixteen had a stage-3 lesion (one with no clearly defined border). RESULTS: Of the forty patients, thirty-four had curettage with use of a high-speed burr. Of these thirty-four, twenty-two had filling of the defect with a cancellous autogenous graft; four, with a cancellous allograft; and three, with polymethylmethacrylate. In five patients, no material was put into the defect. The remaining six patients had resection through the margin of the lesion. Four (12 percent) of the thirty-four patients who had curettage had a local recurrence. No patient who had an excision through the margin of the lesion had a local recurrence. All local recurrences were in skeletally immature girls who were three, four, ten, and eleven years old. Univariate analysis with use of the chi-square, Fisher exact, and Wilcoxon log-rank tests showed that local recurrence was associated only with a young age (p = 0.0036) and open growth plates (p = 0.039). All local recurrences occurred within two years postoperatively, at two, seven, nine, and twenty-four months, and all were treated successfully with a second operation. CONCLUSIONS: Rates of local control of almost 90 percent can be achieved with thorough curettage with use of a mechanical burr and without use of liquid nitrogen, phenol, or other adjuvants in patients who have an aneurysmal bone cyst of an extremity. A young age and open growth plates are associated with an increased risk of local recurrence.

Placenta previa percreta involving the left broad ligament and cervix. A case report.

BACKGROUND: Placenta previa percreta with invasion of the broad ligament and uterine cervix is an extremely rare condition and carries high maternal and fetal morbidity and mortality. CASE: A 39-year-old, multiparous woman with two previous cesarean sections presented in active labor at term with placenta previa percreta involving the left broad ligament and cervix. The patient was managed by antepartum diagnosis of placenta previa accreta, supracervical hysterectomy, and blood transfusion. CONCLUSION: This case was managed consistent with the literature, and favorable maternal and fetal outcomes were achieved.

Oncological outcomes of operative treatment of subcutaneous soft-tissue sarcomas of the extremities.

We reviewed the cases of sixty-two patients who had had a subcutaneous sarcoma to determine the effect of tumor and treatment-related variables on the rates of survival and local recurrence. Fifty-nine (95 per cent) of the patients had had an operation at another hospital before being referred to us. Twenty-nine (47 per cent) of the sixty-two tumors were high-grade, forty-two (68 per cent) were small (five centimeters or less), and thirty (48 per cent) were malignant fibrous histiocytomas. We followed a treatment strategy that consisted of repeat excision with the goal of obtaining wide margins. Excluding thirteen patients who had had a palpable local recurrence at the time of presentation, twenty (49 per cent) of forty-one patients who had had a marginal excision at another hospital had microscopic residual tumor on repeat excision. At a median of fifty-six months after the repeat excision, fifty (81 per cent) of the sixty-two patients had been continuously disease-free, one had no evidence of disease, eight had died of the disease, and three had died of other causes. The five-year rate of disease-free survival was 85 per cent (fifty-three of sixty-two patients). There were three local recurrences, all in patients who had had a marginal resection. No recurrences were noted in patients who had had a wide local excision of the tumor or of the previous operative field. Multivariate analysis revealed that a large tumor (greater than five centimeters), a marginal excision, and adjuvant radiation therapy were associated with a worse prognosis. Excellent rates of survival for patients who have a subcutaneous sarcoma, including those who have a large or high-grade tumor and those who have residual tumor following a previous operation, can be obtained with carefully planned operative treatment alone. We recommend operative excision or repeat excision with wide margins because of the high prevalence of residual tumor. Size is the most important tumor-related factor, and the operative margin is the most important treatment-related factor. The additional value of adjuvant radiation therapy remains unproved.

Prevention of autoimmune diabetes by treatment with anti-LFA-1 and anti-ICAM-1 monoclonal antibodies.

Insulin-dependent diabetes mellitus (IDDM) in humans and mouse models is caused by a T cell-mediated destruction of the beta cells in the islets of Langerhans. The interaction between T cells and their antigens or targets is assisted by adhesion molecules on the surfaces of lymphocytes and counterreceptors on antigen-presenting or other cells. One such pair of molecules thought to be of importance in IDDM is lymphocyte function-related antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) because culture of islet cells with cytokines results in the expression of ICAM-1 on islet cells in vitro. To understand the importance of this interaction in the development of autoimmune diabetes, we have studied the ability of monoclonal antibodies (mAbs) against LFA-1 and/or ICAM-1 to prevent disease in multidose streptozotocin-induced diabetes mellitus (MDSDM). Treatment with both anti-LFA-1 and anti-ICAM-1 mAbs reduced the development of hyperglycemia and insulitis in this model. Treatment with the anti-LFA-1 and anti-ICAM-1 mAbs caused modulation of LFA-1 and ICAM-1 expression on splenocytes, respectively. In mice that were undergoing streptozotocin-induced diabetes, ICAM-1 was found on capillary endothelial cells and on cells surrounding the islets but was not expressed at detectable levels on islet endocrine cells. Thus, interaction between ICAM-1 and LFA-1 is involved in the development of autoimmune diabetes in MDSDM. It is unlikely that ICAM-1 molecules mediate adherence between T cells and islet cells themselves. Our results suggest that treatment with anti-ICAM-1 and anti-LFA-1 mAbs prevents localization of T cells to the islets rather than causing functional impairments of the lymphocytes. The actual sites of interaction between the T cells involved in MDSDM and ICAM-1 are unknown, but may occur on cells that normally express ICAM-1 in the islet or may occur outside the islet itself.

An immunohistochemical study of thyroid Hurthle cells and their neoplasms: the roles of S-100 and HMB-45 proteins.

The histological and cytological features of follicular thyroid neoplasms with oxyphilic change (Hurthle cell tumors) may lead to the differential diagnosis of metastatic malignant melanoma. S-100 and HMB-45 staining was studied in 18 Hurthle cell tumors, 6 Hurthle cell carcinomas, and 5 cases of Hashimoto's thyroiditis using a rabbit polyclonal antibody to S-100 protein, a mouse monoclonal antibody to HMB-45 protein, and the avidin alkaline phosphatase method. All 6 carcinomas and 17 of 18 Hurthle cell tumors exhibited strong cytoplasmic and nuclear staining for S-100. One Hurthle cell carcinoma also contained a spindle cell anaplastic area that was negative for S-100. All cases of Hashimoto's thyroiditis displayed intense staining in Hurthle cells for S-100, with weak to negative staining in nonoxyphilic follicular epithelium. In the three studied lesions, all cases were negative for HMB-45 protein. Three nonthyroid oncocytic tumors (renal oncocytoma, oncocytic carcinoma of the parotid, and parathyroid oxyphilic adenoma) were found to be negative for both S-100 and HMB-45 staining. Hurthle cell lesions should be included in the differential diagnosis of S-100-positive tumors. HMB-45 remains a marker more restricted to melanocytic lesions.

Tumors in hepatobiliary tract and pancreatic islet tissues of transgenic mice harboring gastrin simian virus 40 large tumor antigen fusion gene.

Gastrin is expressed in the gastric antrum and in fetal pancreatic islets but not in adult islets. We have now identified the hepatobiliary tract as another, previously unknown, potential site of gastrin gene expression. Two human gastrin simian virus 40 large tumor antigen (SV40 T antigen) fusion genes containing 1.5 kb of 5' flanking sequence and 10.5 kb that included 5.5 kb upstream, 1.5 kb downstream, and the entire transcribed region were used to generate transgenic mice. Analysis of several transgenic lines, derived from both fusion genes, revealed development of transmissible hepatobiliary tract tumors and pancreatic islet cell tumors. Analysis of each of the tumor cells demonstrates expression of SV40 T antigen but no expression of gastrin. Of the two fusion genes, only the 10.5-kb sequence induces hyperplasia of gastrin-producing cells in the antrum. Analysis of these cells demonstrates expression of SV40 T antigen and gastrin, suggesting that the 10.5-kb sequence is sufficient for gastrin cell hyperplasia in the antrum. These data raise the possibility that gastrin is transiently expressed in the hepatobiliary tract.

Insulin-degrading enzyme is differentially expressed and developmentally regulated in various rat tissues.

Insulin-degrading enzyme (IDE), a cytosolic metalloendoprotease, can degrade insulin, insulin-like growth factor-II, insulin-like growth factor-I, and transforming growth factor-alpha. While IDE has been implicated in the cellular degradation of insulin, other physiological functions of this enzyme are not known. To assess the possible role of IDE in cellular growth and development, we determined the tissue and developmental distribution of the enzyme. Rat IDE cDNA fragments and antibodies directed against human IDE were used to probe IDE transcripts and proteins in rat tissues. The results demonstrate that IDE transcripts are ubiquitous in rat tissues. The level of rIDE transcripts is high in adult rat testis, tongue, and brain; moderate in kidney, prostate, heart, muscle, liver, intestine, and skin; and low in spleen, lung, thymus, and uterus. The sizes of the major transcripts of rIDE are 3.4 and 6.3 kilobases in all tissues analyzed, except testis. Surprisingly, the highest level of rIDE mRNA in the adult rat was in the testis, and the major transcripts of rIDE in this tissue were shifted in size to 3.8 and 6.7 kilobases. Immunocytochemical analysis localized the rIDE mainly in the epithelium of prostate gland and kidney, and the cytosol of liver hepatocytes. During rat development from 6-7 days of age to adulthood, rIDE mRNA levels increased in brain, testis, and tongue; decreased in muscle and skin; and did not significantly change in other tissues examined. These studies reveal regulation of IDE or IDE-related genes in rat tissues and during rat development, suggesting that this enzyme may have multiple functions relating to cellular growth and development.

Induction of tolerance to autoimmune diabetes with islet antigens.

The development of T cell tolerance to self-antigens is imparted principally through negative selection events during thymic ontogeny. However, this tolerance may be limited to antigens that are expressed in the thymus, and additional mechanisms are probably required to regulate autoimmune responses to tissue-specific antigens. Autoimmune diabetes can be induced experimentally by treating susceptible stains of mice with multiple low doses of streptozotocin (STZ). In this report we show that transplantation of isolated islets of Langerhans into the thymuses of adult C57BL/KsJ mice will induce tolerance to the subsequent induction of autoimmune diabetes. This tolerance is tissue specific and thymus dependent. It was not induced by thymic transfer of adrenal tissue or by kidney transfer of islets. Furthermore, depletion of mature T cells was required and the tolerant state was abrogated by the adoptive transfer of normal splenocytes. It is interesting that pretreatment of the islets with STZ enhanced their ability to induce tolerance, and suggests that antigen shedding induced by tissue damage may facilitate transfer of islet antigens to tolerizing cells in the thymus. These findings indicate that thymic tolerance specific for tissue can be stimulated to occur in the presence of atopic tissue-specific intrathymic antigens. Elimination of disease-related T cells in the absence of global immunosuppression represents a novel approach for the prevention of autoimmune disease.

Proinsulin processing by the subtilisin-related proprotein convertases furin, PC2, and PC3.

Experiments using recombinant vaccinia viruses expressing rat proinsulin I coinfected into COS-7 cells with recombinant vaccinia virus expressing human furin, human PC2, mouse PC3 (subtilisin-related proprotein convertases 1-3, respectively), or yeast Kex2 indicate that in this system both Kex2 and furin produce mature insulin, whereas PC2 selectively cleaves proinsulin at the C-peptide-A-chain junction. This is a property consistent with its probable identity with the rat insulinoma granule type II proinsulin processing activity as described by Davidson et al. [Davidson, H. W., Rhodes, C. J. & Hutton, J. C. (1988) Nature (London) 333, 93-96]. PC3 generates mature insulin but cleaves preferentially at the proinsulin B-chain-C-peptide junction. This pattern of cleavage by PC3 is similar, but not identical, to that of the highly B-chain-C-peptide junction-selective type I activity as described by Davidson et al., perhaps due to the presence of a P4 arginine residue near the C-peptide-A-chain junction unique to the rat proinsulins. These results along with data presented on the expression of both PC2 and PC3 in islet beta cells strongly support the conclusion that these proteases are involved in the conversion of proinsulin to insulin in vivo.

Treatment of medulloblastoma in adults.

Seventeen adult patients with medulloblastoma were treated at Rush-Presbyterian-St. Luke's Medical Center and affiliated hospital between 1969 and 1986. All patients had a surgical procedure (total excision in seven patients, partial resection in nine patients, and biopsy alone in one patient) followed by radiation therapy to the craniospinal axis. The 5-year actuarial survival rate is 77% with a disease-free survival of 58%. Five patients have relapsed in the posterior fossa, one in the brain parenchyma, and two in osseous sites. Two of the local relapses occurred more than 4 years after initial treatment. Patients undergoing "total" resection of the tumor fared better than those with partial resection or biopsy only. Local failure was uncommon with posterior fossa doses greater than 55 Gy, and there was a trend toward better local control when the radiation therapy was completed in less than 7 weeks. The histologic indicators of poor outcome were necrosis, high mitotic index, and "classical" histologic appearance.

Prevention of autoimmune diabetes with nonactivating anti-CD3 monoclonal antibody.

Autoreactive T cells mediate diabetes in animal models of insulin-dependent diabetes mellitus (IDDM) and are believed to cause the disease in humans. Therefore, immunotherapies directed against T cells are of particular interest for the treatment of IDDM. One candidate for such immunotherapy is anti-CD3 monoclonal antibodies (MoAbs), but clinical side effects are common with anti-CD3 treatment due to the ability of these MoAbs to activate T cells in vivo. However, F(ab')2 fragments of anti-CD3 are nonactivating and immunosuppressive. We evaluated the effects of whole anti-CD3 MoAb and F(ab')2 fragments in the setting of experimental autoimmune diabetes. Treatment with whole MoAb or F(ab')2 fragments significantly reduced the hyperglycemia induced with multiple low dosages of streptozocin (MDSDM; 232 +/- 23 mg/dl, P less than 0.01 and 235 +/- 16 mg/dl, P less than 0.01 vs. 325 +/- 25 mg/dl, respectively) in male CD1 mice. Both whole MoAb and F(ab')2 fragments suppressed the development of insulitis (P less than 0.001). Treatment with whole MoAb resulted in marked weight loss (10.4 +/- 1.5% of total body wt), and the mice appeared ill and listless, whereas, mice treated with F(ab')2 fragments gained weight (4.9 +/- 5.5% of total body wt) and appeared healthy. Treatment with whole MoAb caused activation of T cells in vivo as reflected by proliferation of freshly isolated spleen cells to recombinant interleukin-2. Depletion of T cells with whole MoAb was more pronounced than with F(ab')2 fragments, and T-cell receptor (TCR) reexpression on remaining cells occurred with F(ab')2 fragments within 48 h after F(ab')2 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)