A vascular model of Tsc1 deficiency accelerates renal tumor formation with accompanying hemangiosarcomas.

UNLABELLED: Tuberous sclerosis complex (TSC) is an autosomal disease caused by inactivating mutations in either of the tumor suppressor genes TSC1 or TSC2. TSC-associated tumor growth is present in multiple tissues and organs including brain, kidney, liver, heart, lungs, and skin. In the kidney, TSC angiomyolipomas have aberrant vascular structures with abnormal endothelial cells, suggesting a role for endothelial mTORC1 function. In the current report, a genetically engineered mouse model (GEMM) with a conditional knockout allele of Tsc1 with a Darpp32-Cre allele displayed accelerated formation of both kidney cystadenomas and paw hemangiosarcomas. All mutant mice developed hemangiosarcomas on multiple paws by 6 weeks of age. By 16 weeks of age, the average mutant hind paw was 4.0 mm in diameter, nearly double the size of control mice. Furthermore, the hemangiosarcomas and kidney cystadenomas were responsive to intraperitoneal rapamycin treatment. Immunoblotting and immunostaining for phospho-S6 (pS6) and phospho-CAD showed that the effect of rapamycin on tumor size was through inhibition of the mTOR signaling pathway. Finally, elevated VEGF mRNA levels were also observed in hemangiosarcoma specimens. Because paw hemangiosarcomas are easily detectable and scorable for size and growth, this novel mouse model enables accelerated in vivo drug testing for therapies of TSC-related tumors. IMPLICATIONS: These findings provide a strong rationale for simultaneous use of this conditional knockout mouse as an in vivo genetic model while seeking new cancer therapies for TSC-related tumors.

Equivalent benefit of rapamycin and a potent mTOR ATP-competitive inhibitor, MLN0128 (INK128), in a mouse model of tuberous sclerosis.

Tuberous sclerosis complex (TSC) is a hamartoma syndrome in which brain, renal, and lung tumors develop and cause both morbidity and death. Loss of either TSC1 or TSC2 in TSC hamartomas leads to activation of mTORC1. Rapamycin and related drugs have been shown to have clinical benefit for these tumors in patients with TSC and those with sporadic forms of TSC-related neoplasms. However, lifelong therapy seems to be required, as tumors are not eliminated by this treatment. We examined the potential benefit of MLN0128, a novel potent mTOR ATP-competitive inhibitor, as a therapeutic strategy for renal cystadenomas that develop in A/J Tsc2(+/-) mice. Rapamycin given by intraperitoneal injection at 3 mg/kg 3 times per week, and MLN0128 given by gavage at 0.75 mg/kg 5 times per week had equivalent effects in suppressing tumor development during a 4-week treatment period, with an approximate 99% reduction in microscopic tumor cell volume. Marked reduction in activation of mTOR complex (mTORC)1 and blockade of cell growth was seen with both drugs, whereas only MLN0128 treatment had effects in blocking mTORC2 and 4EBP1 phosphorylation. However, when either drug was discontinued and mice were observed for two additional months, there was dramatic recovery of tumor growth, with extensive proliferation. Hence, longlasting tumor growth control is not achieved with transient treatment with either drug, and MLN0128 and rapamycin have equivalent therapeutic benefit in this mouse model. Differences in side-effect profiles might make MLN0128 more attractive for treatment of patients with TSC-related tumors, but will require additional study in humans.

Comparison of three rapamycin dosing schedules in A/J Tsc2+/- mice and improved survival with angiogenesis inhibitor or asparaginase treatment in mice with subcutaneous tuberous sclerosis related tumors.

BACKGROUND: Tuberous Sclerosis Complex (TSC) is an autosomal dominant tumor disorder characterized by the growth of hamartomas in various organs including the kidney, brain, skin, lungs, and heart. Rapamycin has been shown to reduce the size of kidney angiomyolipomas associated with TSC; however, tumor regression is incomplete and kidney angiomyolipomas regrow after cessation of treatment. Mouse models of TSC2 related tumors are useful for evaluating new approaches to drug therapy for TSC. METHODS: In cohorts of Tsc2+/- mice, we compared kidney cystadenoma severity in A/J and C57BL/6 mouse strains at both 9 and 12 months of age. We also investigated age related kidney tumor progression and compared three different rapamycin treatment schedules in cohorts of A/J Tsc2+/- mice. In addition, we used nude mice bearing Tsc2-/- subcutaneous tumors to evaluate the therapeutic utility of sunitinib, bevacizumab, vincristine, and asparaginase. RESULTS: TSC related kidney disease severity is 5-10 fold higher in A/J Tsc2+/- mice compared with C57BL/6 Tsc2+/- mice. Similar to kidney angiomyolipomas associated with TSC, the severity of kidney cystadenomas increases with age in A/J Tsc2+/- mice. When rapamycin dosing schedules were compared in A/J Tsc2+/- cohorts, we observed a 66% reduction in kidney tumor burden in mice treated daily for 4 weeks, an 82% reduction in mice treated daily for 4 weeks followed by weekly for 8 weeks, and an 81% reduction in mice treated weekly for 12 weeks. In the Tsc2-/- subcutaneous tumor mouse model, vincristine is not effective, but angiogenesis inhibitors (sunitinib and bevacizumab) and asparaginase are effective as single agents. However, these drugs are not as effective as rapamycin in that they increased median survival only by 24-27%, while rapamycin increased median survival by 173%. CONCLUSIONS: Our results indicate that the A/J Tsc2+/- mouse model is an improved, higher through-put mouse model for future TSC preclinical studies. The rapamycin dosing comparison study indicates that the duration of rapamycin treatment is more important than dose intensity. We also found that angiogenesis inhibitors and asparaginase reduce tumor growth in a TSC2 tumor mouse model and although these drugs are not as effective as rapamycin, these drug classes may have some therapeutic potential in the treatment of TSC related tumors.

Renal and liver tumors in Tsc2(+/-) mice, a model of tuberous sclerosis complex, do not respond to treatment with atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor.

Inactivating mutations of the tumor suppressor gene TSC2 are associated with tumorigenesis in tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis. Statins, as 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, have the potential to limit the growth of these tumors by limiting the isoprenylation of activated GTPases in Tsc2-null cells. We tested atorvastatin as a therapy for (a) ethylnitrosourea (ENU)-enhanced renal cystadenoma and (b) spontaneous liver hemangioma in 129Sv/Jae Tsc2(+/-) mice. ENU-treated Tsc2(+/-) mice were given atorvastatin chow (0.1%, w/w) for 1 or 3 months before sacrifice at 6 months; 129Sv/Jae Tsc2(+/-) mice were given atorvastatin chow (0.1%, w/w) for 6 months before sacrifice at 12 months. All treatment groups were compared with mice of identical genotype and strain background that were fed control chow. Pathologic analyses revealed a predominance of renal cystadenoma in ENU-treated and liver hemangioma in non-ENU-treated 129Sv/Jae Tsc2(+/-) mice. In both cohorts, serum cholesterol levels and levels of phosphorylated S6 and GTP-RhoA in healthy tissue were significantly (>50%) reduced in atorvastatin-treated mice as compared with controls. Following atorvastatin treatment, no significant reduction in tumor size, morphology, or phosphorylated S6 levels was observed for either ENU-associated renal cystadenoma or spontaneous liver hemangioma as compared with the untreated groups. In conclusion, although the marked reduction in cholesterol levels indicates that atorvastatin was effective as an 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, it did not inhibit the growth of tumors that develop in these Tsc2(+/-) models, suggesting that it is unlikely to have benefit as a single-agent therapy for TSC-associated tumors.

Rapamycin weekly maintenance dosing and the potential efficacy of combination sorafenib plus rapamycin but not atorvastatin or doxycycline in tuberous sclerosis preclinical models.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant tumor suppressor syndrome, characterized by hamartomatous growths in the brain, skin, kidneys, lungs, and heart, which lead to significant morbidity. TSC is caused by mutations in the TSC1 or TSC2 genes, whose products, hamartin and tuberin, form a tumor suppressor complex that regulates the PI3K/Akt/mTOR pathway. Early clinical trials show that TSC-related kidney tumors (angiomyolipomas) regress when treated with the mammalian target of rapamycin (mTOR) inhibitor, rapamycin (also known as sirolimus). Although side effects are tolerable, responses are incomplete, and tumor regrowth is common when rapamycin is stopped. Strategies for future clinical trials may include the investigation of longer treatment duration and combination therapy of other effective drug classes. RESULTS: Here, we examine the efficacy of a prolonged maintenance dose of rapamycin in Tsc2+/- mice with TSC-related kidney tumors. Cohorts were treated with rapamycin alone or in combination with interferon-gamma (IFN-g). The schedule of rapamycin included one month of daily doses before and after five months of weekly doses. We observed a 94.5% reduction in kidney tumor burden in Tsc2+/- mice treated (part one) daily with rapamycin (8 mg/kg) at 6 months

Giant multilocular cystadenoma of the prostate responsive to GnRH antagonists.

Giant multilocular cystadenoma of the prostate is a rare, benign, but locally recurrent, tumor that is usually treated by surgery. We report a case initially treated by surgical resection and followed by evaluation of serum prostate-specific antigen values. After evidence of biochemical failure, pathologic recurrence was confirmed and treated by the gonadotropin-releasing hormone antagonist Lupron, with excellent results. The patient was stable without biochemical or radiologic evidence of progression during the last 2.5 years. This result offers a new treatment option for patients with this rare tumor.

Modulation of melphalan and cisplatin cytotoxicity in human ovarian cancer cells resistant to alkylating drugs.

We investigated the effect of pharmacological modulators on the cytotoxic activity of melphalan and cisplatin in human ovarian cystadenocarcinoma cells sensitive (OAW42) or resistant (OAW42MER) to bifunctional alkylating agents. By filter elution experiments we observed a reduced accumulation and a faster repair of melphalan-induced DNA interstrand cross-links in the OAW42MER resistant cells than in the OAW42 parental, sensitive cells. Moreover, resistant cells were characterized by an increased level of mRNA encoding enzymes involved in the nucleotide excision repair pathway, such as ERCC (excision repair cross complementing)1 and ERCC2. Among the modulators used, the topoisomerase I inhibitor topotecan was able to increase melphalan cytotoxic activity in sensitive and resistant cell lines. Topotecan also positively modulated cisplatin activity, although to a variable extent in the two cell lines, as a function of treatment schedule. The energolytic compound lonidamine markedly enhanced the cytotoxicity of melphalan and cisplatin, with a potentiating effect in the OAW42MER resistant cells almost 2-fold that of in the OAW42 sensitive cells. No significant potentiation was observed by using calcium channel blockers, such as verapamil and nimodipine. Conversely, an increase in melphalan cytotoxic activity was determined by flunarizine in OAW42MER resistant cells and, to a lesser extent, in OAW42 sensitive cells. However, the calcium blocker failed to modulate cisplatin activity in both cell lines.

[Studies on serum protein fractions of patients with maxillary sinus cancer undergoing a combination of radiotherapy and chemotherapy: II. Relationship between changes in serum protein fractions and prognosis].

We examined the correlations between changes in serum protein fractions and the prognosis of the patients. The levels of 21 protein components of the sera of 36 patients with maxillary sinus cancer were determined by a single radial immunodiffusion method before and after radiation therapy. The patients with maxillary sinus cancer were treated with combined intra-arterial infusion of bleomycin and external irradiation of 60 Co gamma-rays, and were concurrently treated with 5-fluorouracil at 200 mg/day p.o. The levels of the same protein components were also measured in 34 normal adult as a control. All patients were observed 5 years and 12 years after radiation therapy. In patients who had survived at least 5 years after radiation therapy, the Alb, Tf, Hx, IgG and IgM levels measured before radiation therapy were elevated significantly compared with those who had died within 5 years. In those who had survived at least 5 years, the Alb, Tf, Hx, IgG, IgM, IgA and I alpha I levels measured after radiation therapy were elevated significantly compared with those who had died within 5 years, and AT III was reduced. In cases of maxillary sinus cancer following a period of 5 to 12 years after radiation therapy, multiple regression analysis was used to determine whether increased concentrations of serum protein fractions were associated with good prognosis for the original disease, alpha 2HS. IgM, HX, alpha 1AT and alpha 1X before radiation therapy were positively correlated with survival, whereas AT III, Pmg, Cp, IgA, and alpha 1AG showed negative correlations. After radiation therapy, Pmg, Hx, Cp, Cl inh and Fib were found to be positive factors of survival rate, whereas alpha 2M, alpha 2Pl, I alpha 1, IgA, alpha 1AG and C3 were negative factors.

Natural killer cell activity and progression-free survival in ovarian cancer.

A longitudinal evaluation of the natural killer (NK) cell activity of peripheral blood lymphocytes was performed in 17 patients with advanced ovarian cancer (treated surgically and with subsequent multiagent chemotherapy) for a median follow-up period of 32 months. At the time of primary treatment, the mean value of NK cell activity was significantly lower in patients who then had disease progression (p < 0.05) than in patients with progression-free survival. During the follow-up period, no significant modifications of the NK cell activity were observed; only at the time of clinical disease progression did the NK cell activity show a significant reduction. We conclude that the NK cell activity is a potentially important prognostic factor.

Glutathione S-transferase activity and isoenzyme composition in benign ovarian tumours, untreated malignant ovarian tumours, and malignant ovarian tumours after platinum/cyclophosphamide chemotherapy.

Glutathione S-transferase (GST) isoenzyme composition, isoenzyme quantities and enzymatic activity were investigated in benign (n = 4) ovarian tumours and malignant ovarian tumours, before (n = 20) and after (n = 16) chemotherapy. Enzymatic activity of GST in cytosols was measured by determining 1-chloro-2,4-dinitrobenzene conjugation with glutathione, cytosolic GST subunits were determined by wide pore reversed phase HPLC, using a S-hexylglutathione-agarose affinity column, and isoelectric focussing. Both GST activity and GST pi amount were not related to histopathologic type, differentiation grade, or tumour volume index in untreated malignant tumours. GST isoenzyme patterns were identical in benign tumours and malignant tumours before and after platinum/cyclophosphamide chemotherapy, while GST pi was the predominant transferase. Mean GST activity and GST pi amount were decreased (P < 0.05) in malignant ovarian tumours after platinum/cyclophosphamide chemotherapy compared to untreated ovarian malignant tumours. No relation was found in untreated ovarian tumours between GST pi amount and response to platinum/cyclophosphamide chemotherapy. Thus, within the limitations of the current study no arguments were found for a role of GST in in vivo drug resistance of malignant ovarian tumours to platinum/cyclophosphamide chemotherapy.

P-glycoprotein expression and DNA topoisomerase I and II activity in benign tumors of the ovary and in malignant tumors of the ovary, before and after platinum/cyclophosphamide chemotherapy.

P-glycoprotein (P-gp) expression and DNA topoisomerase (Topo) II are important variables in multidrug resistant tumor cell lines. The aim of this study was to evaluate P-gp expression and Topo I and II activity in benign and malignant epithelial ovarian tumors. P-gp expression was analyzed immunohistochemically in cryostat sections of fresh tumor specimens. In the same specimens Topo I and II activity were measured by, respectively, relaxation of supercoiled plasmid pBR322 DNA and decatenation of kinetoplast DNA. P-gp expression (range, 5-100% positive staining cells) was found in 3 of 6 cystadenomas, 0 of 2 borderline tumors, 15 of 21 untreated ovarian cancers, and 8 of 13 platinum/cyclophosphamide treated ovarian cancers. Median Topo I and II activity were elevated in malignant ovarian tumors compared to benign and borderline tumors. No difference was found between median Topo I activity in untreated ovarian cancer and platinum/cyclophosphamide treated ovarian cancer. High Topo II activity (greater than or equal to 8 x 10(2) units/mg protein) was more frequent in untreated compared to platinum/cyclophosphamide treated samples. Respectively, 8- and 16-fold differences in Topo I and II activity were found in the malignant tumors. Topo II activity in malignant tumors correlated with Topo I activity (r = 0.36, P less than 0.05) and the tumor volume index (r = 0.35, P less than 0.05). However, this last weak correlation cannot explain the 16-fold differences in Topo II activity in malignant tumors. Mitotic index and P-gp expression did not correlate with Topo I or II activity. A large variability in P-gp expression and Topo I and II activity was observed in patients with ovarian cancer.

The consequences of unsuspected secondary ovarian neoplasia: a clinical presentation of four case histories.

Secondary ovarian neoplasia is a common clinical entity which represents 3 to 8% of all ovarian tumors. However, despite its apparent prevalence, it remains a diagnostic and management dilemma. The ability to differentiate primary ovarian carcinoma from metastatic disease to the ovary has significant therapeutic and prognostic implications. Four case histories are presented which will demonstrate the necessity of formulating a complete differential diagnosis as well as the need for a thorough preoperative bowel examination.

[High-dose CDDP therapy using a balloon-occluded arterial infusion (BOAI) in recurrent ovarian cancer].

An intraarterial high-dose CDDP, using a balloon catheter, has been infused into five patients with a recurrent ovarian cancer. The dose of CDDP was 150 mg-200 mg/body. Two patients experienced a partial tumor regression, whereas three showed no change. With regard to the toxicity, the NAG index (NAG activity/urinary creatinine) rose markedly in all patients, but toxicity was almost similar to that seen from a systemic administration. No significant catheter complication occurred.

Malignant clear cell cystadenoma of the ovary.

A 50-year-old female had suffered from abdominal distension and a lower abdominal mass for 3 months. Ultrasonographic examination demonstrated a myoma uteri pattern with a large cystic ovarian tumor. Laparotomy was done on August 30, 1983 and simple total hysterectomy with bilateral salpingo-oophorectomy was carried out. FIGO stage IIC ovarian cancer was stated clinically. Histologically, clear cell cystadenoma of right ovary having benign, proliferative and malignant epithelial elements without dense ovarian stroma was found and diagnosed as malignant clear cell cystadenoma. In spite of Cisplatin therapy, the patient expired on April 6, 1984.

[An experimental study of cisplatin and it's antidote, sodium thiosulfate, in ovarian pseudomyxoma implanted into nude mice abdominal cavity].

While Cisplatin was found to be effective in treating pseudomyxoma, its side effects, particularly on the kidneys, were very strong. Recently a "two channel therapy", in which it is used along with sodiumthiosulfate, has been reported to minimize the side effects. The authors used nude mice with pseudomyxoma implanted into the abdominal cavity in an experiment in which Cisplatin was introduced into the abdominal cavity and sodiumthiosulfate injected subcutaneously in a two channel procedure to determine the effect of sodiumthiosulfate on the action of Cisplatin on the pseudomyxoma and on the side effects of Cisplatin. The following results were obtained: The survival rate was improved when sodiumthiosulfate was administered with 40 mg/kg and 20 mg/kg of Cisplatin. Body weight loss was controlled when sodiumthiosulfate was administered with 20 mg/kg of Cisplatin. On the other hand, the histological effect of Cisplatin was also suppressed when sodiumthiosulfate was co-administered. In this study, we failed to observed any histological changes in the kidneys and liver attributable to Cisplatin.

In vivo chemosensitivity testing in patients with gynecologic malignancies and nude mouse xenografts by monitoring cell kinetic parameters and DNA distribution patterns: a preliminary report.

Using flow cytometry (FCM) and autoradiography we have evaluated changes in DNA-ploidy patterns as well as cell-cycle perturbations after chemotherapy in 12 patients with accessible tumors and 22 nude mice xenografts. Gynecologic malignancies growing as nude mouse xenografts serve as an experimental model to study the effect of chemotherapy at the cellular level. Excellent correlation was found between fine needle aspiration (FNA) and biopsy material for either FCM or autoradiography. It now appears possible to study the effects of chemotherapy in the patient as well as the animal model using serial FNAs as a microsampling technique.

[Clinical studies on malignant ovarian tumor--with special reference to therapy and prognosis of epithelial ovarian tumor].

Two hundred and ninety-two (75%) patients with common "epithelial" tumors of 391 patients with various kinds of malignant ovarian tumors treated at our hospital and other 14 hospitals between 1971 and 1981 were evaluated particularly to determine 1) the prognostic value of the disease stage and 2) results of therapy. Of the 292 patients, 49.8% were classified in Stage I, 12.8% in Stage II, 27.2% in Stage III and 10.1% in Stage IV. The 5-year survival rates for the various stages were: Stage I, 70.2%; Stage II, 50. 0%; Stage III, 3.4%; and Stage IV, 0%. Postoperative chemotherapy was given to 56.9% of the patients. The prognosis of patients with Stage Ia or Ic who received adjuvant chemotherapy improved, as compared to those who were treated with surgery alone. However, the role of chemotherapy in advanced stages (Stage III and IV) remains unclear.