Beppino C. Giovanella

TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal

Malignant cells, like all actively growing cells, must maintain their telomeres, but genetic mechanisms responsible for telomere maintenance in tumors have only recently been discovered. In particular, mutations of the telomere binding proteins alpha thalassemia/mental retardation syndrome X-linked (ATRX) or death-domain associated protein (DAXX) have been shown to underlie a telomere maintenance mechanism not involving telomerase (alternative lengthening of telomeres), and point mutations in the promoter of the telomerase reverse transcriptase (TERT) gene increase telomerase expression and have been shown to occur in melanomas and a small number of other tumors. To further define the tumor types in which this latter mechanism plays a role, we surveyed 1,230 tumors of 60 different types. We found that tumors could be divided into types with low (<15%) and high (≥15%) frequencies of TERT promoter mutations. The nine TERT-high tumor types almost always originated in tissues with relatively low rates of self renewal, including melanomas, liposarcomas, hepatocellular carcinomas, urothelial carcinomas, squamous cell carcinomas of the tongue, medulloblastomas, and subtypes of gliomas (including 83% of primary glioblastoma, the most common brain tumor type). TERT and ATRX mutations were mutually exclusive, suggesting that these two genetic mechanisms confer equivalent selective growth advantages. In addition to their implications for understanding the relationship between telomeres and tumorigenesis, TERT mutations provide a biomarker that may be useful for the early detection of urinary tract and liver tumors and aid in the classification and prognostication of brain tumors.

Heterotransplantation of human cancers into nude mice. A model system for human cancer chemotherapy.

More than 200 human cancers have been successfully transplanted into nude athymic mice in our laboratory. Strict sterile techniques must be applied in caring for these animals and meticulous attention given to the preparation of the surgical specimens. That the tumors remain human in the mice has been documented by isozyme and karyotype analyses. Also, histopathologic comparisons between the original human tumors and their transplanted counterparts in the mice are strikingly similar or identical. Preliminary results of chemotherapy of human cancers in the nude mice are extremely encouraging. At this point, we believe that the greatest potential for the nude mice lies in their use in testing anticancer therapies for human neoplasms without placing the patients at risk. Cancer 42:2269–2281, 1978.

Anticancer exffect of 9-nitrocamptothecin liposome aerosol on human cancer xenografts in nude mice

Purpose: To test the anticancer properties of the water-insoluble derivative of camptothecin, 9-nitrocamptothecin (9-NC) against human breast, colon and lung cancer xenografts in nude mice when administered in liposome aerosol. Methods: The drug was formulated with dilauroylphosphatidylcholine and nebulized in a particle size of 1.6u2009μmu2009±u20092.0 mass median diameter to deliver doses of usually less than 200u2009μg/kg daily, 5 days per week. 9-NC liposome aerosols were generated with a Aerotech II nebulizer (CIS-USA) flowing at 10u2009l/min from a compressed air source and delivered to mice in sealed plastic cages or in a nose-only exposure chamber. Results: Tumor growth was greatly reduced or tumors were undetectable after several weeks of treatment. Colon tumor was least responsive. 9-NC was better than the parent compound, camptothecin, also water-insoluble, tested by aerosol in a similar liposomal preparation. Equivalent doses of 9-NC liposome preparations administered by mouth were substantially without effect while there was some effect, but limited, of the liposome preparation given intramuscularly. Conclusions: 9-NC liposome aerosol was strikingly effective in the treatment of three human cancer xenografts growing subcutaneously over the thorax in nude mice at doses much smaller than those traditionally used in mice administered by other routes.

Correlation between response to chemotherapy of human tumors in patients and in nude mice

Human tumors serially heterotransplanted in nude mice have been tested for their response to chemotherapeutic agents. Fourteen melanomas, 14 colorectal carcinomas, and 14 breast carcinomas have been used. Each tumor originated in a different patient. The tumors were maintained by serial subcutaneous transplantation in nude mice. For the experiments in this study, each neoplasm was transplanted under the kidney capsule of 60 to 100 adult nude mice. The areas of the individual tumor implants were precisely measured immediately after insertion using a stereo microscope equipped with a micrometric ocular. The animals were then divided into groups of six to ten animals each. One group was injected daily with saline and served as controls. The mice in the remaining groups were injected daily for eight days with one of the following chemotherapeutic agents—Adriamycin (doxorubicin), 5‐fluorouracil, methotrexate, Cytoxan (cyclophosphamide), Alkeran (melphalan), vincristine, vinblastine, methyl‐CCNU, or BCNU—at optimum doses (the maximum dose tolerated that causes less than 10% weight loss). Treatment was initiated when the implants were well established, having roughly doubled their initial mass. The animals were then sacrificed and the tumors measured again. A drug was rated effective only if it inhibited growth of the tumor by 99% or more. The results so obtained were compared with the published results of various clinical trials. When the sensitivity of the human tumors in the mice was compared with the sensitivity of tumors of the same type that had been treated in human patients, a close correlation was found. The panel study detected nine of ten effective drugs, giving only two false‐positive results. Our data strongly support the validity of heterotransplants of human tumors in the nude mouse as a predictive system for testing new anticancer agents and in determining optimal treatment schedules and combinations of known drugs.

Phase I Clinical and Pharmacological Studies of 20‐(S)‐Camptothecin and 20‐(S)‐9‐Nitrocamptothecin as Anticancer Agents

Groups of 52 and 29 patients with refractory cancers received either native camptothecin (CPT) or 9-nitrocamptothecin (9NC), respectively, in Phase I clinical trials designed to determine the maximum tolerated dose, toxicity and potential efficacy of orally administered camptothecins. Favorable responses occurred with both compounds (11% after CPT, 24% after 9NC). Although both agents could be taken safely for extended periods, dose limiting toxicities were substantial. Diarrhea was the major clinical problem with CPT, and myelosuppression with 9NC. Both compounds could cause hemorrhagic cystitis. The antitumor activity demonstrated suggests that further investigation of orally administered camptothecin analogs is warranted.

Eleven years' experience with hyperthermic perfusion for melanoma of the extremities.

Since 1967, we have used a system of hyperthermic perfusion with melphalan to treat patients with melanoma of the extremities. This report presents additional follow-up data on 165 patients, first reported on in 1975, who underwent 185 perfusions. The 70 patients classified as stage I (Stehlin classification system) had a 5-year survival rate of 86.3%. The 5-year survival rate for the 73 patients with stage II and stage III disease was 52.5%. There was a dramatic increase in the 5-year survival rate for patients with stage IIIA disease, from 22.2% prior to the use of heat to 74% for 30 patients undergoing hyperthermic perfusion.RésuméDepuis 1967 nous utilisons, pour le traitement des mélanomes des membres, un système de perfusion au melphalan en hyperthermie. Le présent rapport complète une première étude de follow-up présentée en 1975 et portant sur 165 malades qui ont eu 185 perfusions. Les 70 malades classés stade I (classification de Clark-From) ont une survie à 5 ans de 86.3%. Pour les 73 malades aux stades II et III, la survie à 5 ans est de 52.5%. Lamélioration de la survie est spécialement importante pour les malades au stade IIIA: 22.2% avant la perfusion hyperthermique, 74% des 30 cas qui ont bénéficié de cette technique.

Dependence of Anticancer Activity of Camptothecins on Maintaining Their Lactone Function

Abstract: Camptothecins contain a lactone ring that exists in the closed form below ph 7. Above 7, the open (CPT+) and the closed (CPT) form coexist in a 50–50 ratio in mouse plasma and in a 90–10 ratio in human plasma due to the high affinity of human serum albumin (HSA) for CPT+. CPT+ is much less toxic than CPT and it is excreted much faster. In complete RPMI 1640 culture medium, the equilibrium CPT+‐CPT is 50–50. If 4% HSA is added, it moves to 90–10 modeling for the human physiological situation.

9-Nitrocamptothecin Liposome Aerosol Treatment of Human Cancer Subcutaneous Xenografts and Pulmonary Cancer Metastases in Mice

Abstract: The purpose of this study was to test the anticancer properties of the water‐insoluble derivative of camptothecin, 9‐nitrocamptothecin (9NC), administered in a liposome formulation (L‐9NC) in aerosol to mice with subcutaneous xenografts of three human cancers and in mice with murine melanoma and human osteosarcoma pulmonary metastases. The drug was formulated with dilauroylphosphatidylcholine and nebulized in particle sizes of 1.2–1.6 μm mass median aerodynamic diameter and a geometric standard deviation of 2.0. The aerosol was generated with the nebulizer flowing at 10 l/min and delivered to mice in sealed plastic cages or in a nose‐only exposure chamber. Aerosol was administered for 15 min to 2 hr daily, delivering deposited doses in the respiratory tract of 8.1–306.7 mg of 9NC/kg. With subcutaneous tumors, growth was greatly inhibited or tumors were undetectable after several weeks of treatment. We also showed that oral dosage with L‐9NC had no detectable effect on cancer growth, and thus the benefit from aerosol treatment was due to pulmonary deposition and not the larger fraction of drug deposited in the nose of mice during aerosol treatment which is promptly swallowed. Intramuscular L‐9NC in slightly larger doses than given in the aerosol had detectable anticancer activity, but it was significantly less than in mice receiving the drug by aerosol. With metastatic pulmonary cancers, treated animals showed highly significantly less cancer growth than control animals. L‐9NC aerosol showed a major therapeutic benefit in the treatment of subcutaneous human cancer xenografts in nude mice, suggesting that cancers at systemic sites might be responsive to this treatment. In addition, the strong anticancer effect of L‐9NC aerosol on pulmonary metastases offers a therapeutic approach for treatment of pulmonary cancers. Thus, L‐9NC aerosol may have applicability in the treatment of cancers throughout the body.

Effect of excess thymidine on the growth of human melanoma cells transplanted in thymus deficient nude mice

The effect of thymidine (TdR) on the growth of a human melanoma transplanted in nude mice has been studied. It was found that the injection of 1 g/kg/h of TdR for at least 72 h is sufficient to suppress the growth of the melanoma cells. This inhibition lasts for the duration of the treatment, and causes no apparent toxicity to the host. Nude mice treated for 6--9 days with TdR survived 158 days after melanoma transplant versus 126 days for the controls.

Modified Lactone/Carboxylate Salt Equilibria in Vivo by Liposomal Delivery of 9‐Nitro‐Camptothecin

Abstract: The lactone stability of camptothecins is critical for their anticancer activity. A stable liposomal 9‐nitro‐camptothecin formulation was developed to circumvent the drawbacks of low aqueous solubility and lactone instability and to provide sustained release of the agent in blood circulation. The potential merits of the formulation were demonstrated by its profoundly improved lactone stability in vivo, favorable pharmacokinetic and biodistribution characteristics in rats, and enhanced preclinical efficacy in tumor‐bearing athymic mice.