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The retinoids have been investigated extensively as chemopreventive and therapeutic agents in a variety of neoplasms. They have been shown to inhibit the proliferation of transformed cell lines in vitro and transplanted tumors in vivo. In cultured murine melanoma cells, retinoids inhibit proliferation and induce differentiation. Human melanoma cell lines have shown a mixed response. The clinical experience with retinoids in melanoma has been limited. Previously we investigated the activity of topical B-all-trans-retinoic acid (Retin-A, vitamin A acid, retinoic acid, and tretinoin) against intracutaneous metastases from malignant melanoma. We saw complete remission of multiple lesions in one individual and regression of several lesions in a second patient. This experience led us to conduct the present pilot trial of topical tretinoin in dysplastic nevus syndrome. The latter is a precursor of malignant melanoma. We saw regression of some of the treated lesions to benign nevi showing minimal or no dysplasia. Thus topical tretinoin appears to possess some activity against melanoma and at least one of its precursor conditions. In view of these preliminary results, more extensive trials are warranted to better define the role of tretinoin in the chemoprevention of malignant melanoma in high-risk lesions.

Human malignant melanoma can form tumor colonies in soft agar, and at least two major morphological variants can be identified. Host cells play an important role in human melanoma colony formation, and their effects on the colony variants are selective. Neuroendocrine compounds also modulate expression of the melanoma TCFU pigmentary variants. This system appears suitable for evaluation of the response of melanoma TCFUs to chemotherapy, retinoids, heat, and radiation.

The prevention and/or suppression of preneoplastic foci in animals has been convincingly demonstrated with the use of a variety of compounds, including retinoids. We report here a phase I/II trial of beta-trans-retinoic acid delivered via a collagen sponge/diaphragm insert for neoplasia of the cervix. Eighteen patients with biopsy-proved grades 2 and 3 cervical intraepithelial neoplasia underwent four consecutive daily applications of trans-retinoic aicd to the cervix. Conization of the cervix was performed 4 weeks later. Significant vaginal toxicity in 10 of 18 (55%) patients was unrelated to the concentration or carrying media of the drug. A reduction in size of the intraepithelial lesion was noted by colposcopy in six of 18 (33%) patients with complete resolution of disease at conization in two patients. A new delivery system designed to avoid vaginal toxicity while defining the optimal concentration and dosing schedule for these patients is currently under investigation.

Retinoids are effective suppressors of the phenotypic development of cancer in many animal systems, whether the process is initiated by chemical, physical or viral carcinogens. Cases of cervical intraepithelial neoplasia are excellent for studying the effectiveness of retinoids as chemopreventive agents because the process can be closely followed by serial colposcopic and pathologic (cytology or biopsy) means and changes in the condition safely monitored. We have previously conducted a phase I study of trans-retinoic acid (Tretinoin) given topically by a collagen sponge and cervical cap. A dose of 0.372% was selected for phase II trial. We have treated 20 patients with topical retinoic acid, and a complete response with total regression of disease was obtained in 50%. Systemic and cervical side effects were mild and vaginal side effects moderate but tolerable. These results provide a clinical basis for a randomized, double-blind phase III study to definitely answer the question of whether retinoic acid is an effective chemopreventive agent for cervical cancer.

We measured the effect that staining with 2-(P-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) had on the number and size distribution of tumor colonies counted using an optical image analyzer (FAS II). Staining increased the number of tumor colonies counted. By using opaque tumor cells or pigmented melanoma cells and measuring colony growth kinetics, we demonstrated that the use of INT staining to assist in counting tumor colonies artificially increased the size of viable tumor cell aggregates by adding a red precipitate to the outside surface of the cells. Laboratories that are using the INT method for drug screening are probably measuring colonies down to and below 42 microns in diameter. These small colonies could result from as few as one or two divisions. Thus, potentially useful drugs may be missed in the screen because of the presence of abortive colonies: i.e., lethally damaged cells completing only one or two divisions.

The direct effect of continuous exposure to prostaglandins on the cloning efficiency and proliferative capacity of human malignant melanoma colony-forming cells in soft agar was evaluated. Prostaglandin A1 (PGA1) and prostaglandin E1 (PGE1) effected a dose-dependent inhibition of colony formation and proliferative capacity. PGA1 at a concentration of 5 microgram/ml reduced colony formation of cells from human melanoma cell strains C8054, C8130, and C822 by at least 85%. PGA1 also inhibited colony formation of cells obtained directly from biopsies of melanoma tissues from eight patients by greater than 70% at a concentration of 5 microgram/ml. A steep dose-response curve was evident by the little effect of PGA1 on colony formation at a concentration of 0.5 microgram/ml. The mean 50% inhibitory doses for PGA1 and PGE1 were 1.25 and 4.25 microgram/ml, respectively. Prostaglandin A2 was much less effective than PGA1 in inhibiting melanoma colony formation. The related prostaglandins (prostaglandin B1, prostaglandin F1 alpha, and prostaglandin E2 alpha) had little or no effect on colony formation. Overall, these results suggested that the presence of a carbonyl group at position 9 of the cyclopentane ring may be required for inhibitory activity as prostaglandins of the A and E series inhibited human melanoma cell growth. PGA1 and PGE1 did not effect a rise in cyclic adenosine 3':5'-monophosphate levels in C8054 and C8130 cells. However, while alpha-melanocyte-stimulating hormone and prostaglandin F2 alpha did generate a rise in adenosine 3':5'-monophosphate levels in C8054 cells, these hormones had no effect on colony formation. These results are consistent with the notion that the PGA1 and PGE1 inhibition of melanoma colony-forming cells occurs via a noncyclic nucleotide mechanism.

Three human malignant melanoma cell strains (C8146A, C8146C, and C83-2CY), three established human melanoma cell lines (A375P, A375M, and C8161), and one selected human melanoma subline (A375P-5) were studied to determine if invasion through a reconstituted basement membrane-coated filter (RBMF), which selects the most aggressively invasive cells, would also modulate the cloning efficiency of these cells in soft agar. With the use of the Membrane Invasion Culture System (MICS), all cell strains tested showed a significant increase in cloning efficiency (1.05-9.3-fold) following transit through the RBMF when compared to unmanipulated populations. The established cell lines (A375P, A375M, and C8161) and the A375P-5 subline showed either a decrease or unaltered status in cloning efficiency after invasion. However, all cells demonstrated a consistent decrease in clonogenicity following transit through an uncoated filter compared with RBMFs, thus suggesting the influence of the extracellular matrix on tumor cell clonogenic properties. In general, the established cell lines were more clonogenic before invasion of the RBMF compared with the cell strains, and no correlation was found between clonogenic potential and invasive or metastatic capability. These data may provide important insight into the underlying mechanisms of tumor cell invasion and the subsequent formation and dissemination of metastases in vivo.

Purpose: To provide an overview of the physiologic long-term and late effects of adult cancers and cancer treatments by a review of the medical and nursing literature., Data Identification: Primarily from an English-language literature search using MEDLINE (1980 to 1988) and Index Medicus (1980 to 1988)., Study Selection: After a consensus review by four observers, 285 articles were selected that addressed the stated purpose., Data Extraction: Four observers assessed the literature using predetermined criteria for eliciting information about long-term and late effects. RESULTS AND DATA SYNTHESIS: Much has been written about the acute phases of cancer and cancer treatments. In comparison, relatively few data are available that define physiologic long-term: and late effects of cancer treatments in adult survivors. Review of the existing data showed that these sequelae may affect virtually any body system months or years after treatment ends. In addition, few prospective studies dealing with physiologic survivorship issues have been done., Conclusions: Health care providers need to be aware of long-term or late complications that may affect the increasing number of adult cancer survivors. Attention to treatment regimens in the acute cancer phase and careful follow-up once the disease is eradicated may help to prevent or manage these complications. More prospective research should be done in this area.

Definition of survival and measurement of colony size in soft agar assays is important in establishing in vitro radiation survival curves. Conventionally, survival is assessed according to colony-forming ability. The distinction between small colonies that are abortive and those that are viable often involves a difficult and arbitrary choice for the investigator. We have examined the effect of different minimum colony sizes (greater than or equal to 25, greater than or equal to 50, greater than or equal to 75, and greater than or equal to 100 cells) on ionizing radiation survival curves for cells from established murine (CCL 53.1) and human (M1RW5) melanoma cell lines as well as from short-term human melanoma cell strains (C8146A, C8146C, C8161, C83-2C, C82-7A1, and C8442) and patient biopsy (83-4). Single cell suspensions were plated in the upper layer of the agar bilayer and cells were irradiated by single dose X rays. Giant cells did not form in colonies containing 50 or more cells. D0 values were highest (D0 values, from 390 to 100 cGy) for cells forming smaller colonies (greater than or equal to 25 cells, greater than or equal to 4-5 doublings) and lowest (D0 values, from 190 to 50 cGy) for cells forming larger colonies (greater than or equal to 100 cells, greater than or equal to 6-7 doublings). Therefore, apparent radiosensitivity was dependent on colony size selected for analysis. Precise measurement of colony size was important in establishing radiation survival curves because errors in determining the colony size will alter apparent radiosensitivity of cells. These results should help define the biological meaning of tumor colony growth in semisolid medium, and alter the interpretation of survival curves which measure sensitivity to agents using this assay.

Purpose: To address the psychosocial implications of surviving adult cancers by a comprehensive review of the literature., Data Identification: An English-language literature search using MEDLINE (1970 to 1988). Index Medicus (1970 to 1988), and bibliographic reviews of textbooks and review articles., Study Selection: Of 103 originally identified articles, 58 that specifically addressed the stated purpose were selected., Data Extraction: Four authors reviewed and critiqued the literature extrapolating the major themes on this topic., Results of Data Synthesis: There is little information on the many psychosocial variables that affect an adult's long-term cancer survival trajectory. Collation of data identified the following significant psychosocial themes: fear of recurrence and death, relationships with the health care team, adjustment to physical compromise, alterations in customary social support, isolationism, psychosocial reorientation, and employment and insurance problems., Conclusions: The continuation of a rehabilitation effort begun around the initial diagnosis of cancer would be instrumental in providing post-therapy evaluation and guidance needed by adult long-term survivors of cancer. Education, research, and support interventions need to be mobilized for this population of adults with a history of cancer.

A procedure was developed to directly measure the self-renewal capacity of clonogenic cells from biopsies of metastatic human malignant melanoma. A culture of colony-forming cells was performed with bilayer agar in microtiter wells. The number of live tumor cells from biopsies of melanoma tissue was determined and was used to calculate plating efficiencies. Sequential photography showed that cells did not migrate in agar, thereby documenting that all the cells within colonies were direct descendants of clonogenic cells. A calibrated pneumatically controlled micropipet attached to a micromanipulator was used to quantitatively remove melanoma colonies without removing adjacent cells or agar. Plucked primary colonies were mechanically disaggregated into single cells; viability was greater than 95% as determined by trypan blue dye exclusion. Dose-related formation of secondary colonies was observed after replating of cells from pooled primary colonies. Cells from individual colonies were replated, and secondary colonies formed. These techniques allowed a simple and direct assessment of the self-renewal capacity of colony-forming melanoma cells.

A phase II trial was conducted to determine the clinical activity of amsacrine (m-AMSA) in patients with heavily pretreated solid tumors, myeloma, and lymphoma at the University of Arizona Cancer Center. Additionally, m-AMSA was evaluated at other Southwest Oncology Group institutions in breast cancer, myeloma, melanoma, and oat cell cancer of the lung. At a dose of 120 mg/m2 given iv every 28 days, 12 partial responses were observed in 221 patients evaluable for response. Some antitumor activity was observed in breast cancer (four responses of 65 patients), non-Hodgkin's lymphoma (three of nine), Hodgkin's disease (two of five), and sarcoma (two of 15). A partial response was also documented in one of two patients with cervical cancer. Among the 135 patients treated at the University of Arizona who were extensively evaluated for toxic effects, only myelosuppression and anemia were seen in a significant number of patients. At this dose and schedule, 29% of patients developed leukopenia of less than 3000 cells/mm3, 16% developed a thrombocytopenia of less than 100,000 cells/mm3, and 29% had an acute fall in hemoglobin of greater than or equal to 2 g/100 ml. In addition, two patients suffered grand mal seizures which were not clearly drug-related. These results suggest that further study of m-AMSA in lymphoma, sarcoma, and cervical cancer is warranted.

We have identified three distinct groups of patients with metastatic malignant melanoma that differ in their clinical behavior and responsiveness to therapy. These include a favorable group of patients with lymph node or skin metastases, an intermediate prognostic group with lung or bone metastases, and a poor prognostic group with brain or gastrointestinal involvement. These three groups differed in their predicted objective response to therapy, in their likelihood to achieve a complete remission, and in their median survival.

Although newer combined modality approaches, including neoadjuvant cytotoxic chemotherapy, for patients with squamous cell carcinoma (SCCA) of the head and neck have produced high initial complete response rates, they have not improved overall survival for patients with advanced disease. Vitamin A plays an essential role in the normal differentiation of epithelial tissues. Retinoids, analogs of vitamin A, are active in certain premalignant and malignant disorders including SCCA. Six studies, including one recently reported placebo-controlled randomized trial, have demonstrated the efficacy of retinoids in oral leukoplakia. Two studies (totalling 48 patients) have shown significant retinoid activity (67% overall complete response rate) in patients with aggressive, recurrent laryngeal papillomatosis. Two trials (including a randomized phase II trial) of isotretinoin in advanced, refractory SCCA of the head and neck have produced an objective response rate of 16%, which is comparable to that reported in single-agent studies with cytotoxic drugs. There is a need for further study of retinoids in head and neck cancer. The high initial response rates with current therapy and the high subsequent risks of local recurrence and of developing second primary tumors in head and neck cancer patients offer an excellent opportunity to investigate the use of retinoids as adjuvant therapy for this malignancy.

A human platelet sonicate was evaluated for its effects on the growth of human metastatic melanoma colony-forming cells in soft agar from cells in culture and from biopsies. The addition of platelet sonicate increased both cloning efficiency and proliferative capacity in that more and larger colonies were formed. In more detailed studies under growth-limiting conditions, melanoma cellular responses to known growth factors were compared to the activity found in the platelet sonicate. None of the growth factors tested either alone or in combination, including platelet-derived growth factor, epidermal growth factor, alpha-type transforming growth factor, and beta-type transforming growth factor, were capable of inducing melanoma colony formation to the 12-fold stimulation observed with the platelet sonicate. Treatment of platelet sonicate with dithiothreitol, trypsin, or acid resulted in loss of activity for human melanoma. Our results suggest that human platelets contain an acid-sensitive protein which can support the expression of the transformed phenotype of human melanoma, and this factor is distinct from acid-stable activities previously characterized from human platelets.

In this presentation, issues that influenced the development of policies for inclusion of quality of life end points in certain Southwest Oncology Group clinical trials are reviewed. The key policies recommended by us and adopted by the Cancer Control Research Committee of the Southwest Oncology Group are as follows: (a) Begin assessment of quality of life in specific types of phase III protocols. (b) Always measure physical functioning, emotional functioning, symptoms (general and protocol specific), and global quality of life separately. (c) Include measures of social functioning and additional protocol-specific measures if resources permit. (d) Use patient-based questionnaires with psychometric properties that have been documented in published studies. In this review, we also recommend specific questionnaires. Our recommendations may prove useful for other cancer clinical trials groups and for multi-institution trials of treatment for chronic diseases.

A tumorigenic human cell line was derived from a patient with metastatic melanoma. Cells were treated with adriamycin or actinomycin D in order to assess morphological alterations induced by these anticancer agents. Exposure to 0.01 micrograms/ml adriamycin for one hour caused no observable morphological abnormalities as determined by SEM, while 0.1, 1.0 and 10.0 micrograms/ml concentrations of adriamycin produced surface alterations in the form of blebs, filopodia, microvilli and cell rounding. These alterations may be drug-affected changes of the cell surface or may reflect phases of the cell cycle directed by adriamycin action on the nucleus. Cell morphology appeared normal by SEM for 0.01, 0.1, 1.0 and 10.0 micrograms/ml concentrations of adriamycin when the cells were allowed to recover in drug-free media for 24 hours after initial drug incubation. Concentrations of 0.1, 1.0, and 10.0 micrograms/ml actinomycin D for 24 hours created morphological alterations characterized by cell rounding and long, dendritic-like processes. TEM of colonies treated in soft agar for 24 hours with either 1.0 micrograms/ml adriamycin or 1.0 micrograms/ml actinomycin D showed major morphological effects identified by increased cytoplasmic vacuolization and nuclear disintegration.

alpha-Melanocyte-stimulating hormone (MSH) is known to stimulate melanogenesis in murine melanoma, particularly in Cloudman S-91 melanoma cells. The effects of MSH and insulin on the proliferation of S91 murine melanoma cells have aroused controversy; in various reports, both hormones have been reported to either stimulate or inhibit murine melanoma growth. In our studies both MSH and insulin stimulated the colony-forming ability and the proliferative capacity of S-91 murine melanoma cells grown in soft agar with either serum-supplemented or serum-less medium. Unless insulin and/or MSH were present, Cloudman S-91 melanoma cells failed to clone in soft agar. The insulin effect was greater than that of MSH, and was more pronounced in serum-less than in serum-supplemented medium. The concurrent treatment of S91 melanoma cells with both MSH and insulin resulted in a greater increase in the total number of colonies formed than caused by treatment with either hormone alone. The combined MSH-insulin stimulation of anchorage-independent growth was specific, since the effect could not be mimicked by epidermal growth factor (EGF), gonadotropin-releasing hormone (GRH), luteinizing hormone (LH), nerve growth factor (NGF) or platelet-derived growth factor (PDGF). Therefore, MSH and insulin may be specific growth factors for murine melanoma cells.

A potential application of the human tumor stem cell colony assay is to guide Phase II clinical investigations by identifying classes of tumors (or individual patients) which are sensitive in vitro to a new antitumor compound. We have tested human tumor stem cells from 140 tumor biopsies representing 20 different tumor types for chemosensitivity to the Phase II drug 4'-(9-acridinylamino)methanesulfon-m-anisidide. In vitro sensitivity was defined as a reduction in the number of tumor colony-forming cells to 30% of the control or less after a 1-hr exposure to one-tenth of the pharmacologically achievable plasma concentration of 4'-(9-acridinylamino)methanesulfon-m-anisidide. In vitro sensitivity was found in 29 cases: non-Hodgkin's lymphoma (2 of 2); cervical carcinoma (1 of 1); sarcoma (3 of 6); neuroblastoma (1 of 2); acute myelogenous leukemia (6 of 16); chronic myelogenous leukemia (1 of 3); melanoma (8 of 34); uterine carcinoma (1 of 5); lung carcinoma (1 of 9); ovarian carcinoma (4 of 36); and breast carcinoma (1 of 11). Prospective in vitro-in vivo correlations in eight patients with various tumor types showed that three of three patients sensitive in vitro to 4'-(9-acridinylamino)methanesulfon-m-anisidide responded in vivo, while five of five patients resistant in vitro had no clinical response. The results provide support for further evaluation of the utility of the human tumor stem cell colony assay for targeting Phase II clinical trials.

We have used counterflow centrifugal elutriation to separate the nucleated cells of normal human bone marrow into their major morphologically identifiable cellular components. Human bone marrow cells were separated simultaneously into the major cell types--lymphocytes, monocytes, granulocytes, and erythroid precursors. Significant concentration of bone marrow cell types could be achieved, including myeloblasts (x 25), promyelocytes (x 43), early myelocytes ( x 22), late myelocytes (x 17), metamyelocytes (x 6), eosinophils (x 40), basophils (x 55), erythroblasts (x 35), monocytes (x 40), and plasma cells (x 35). Cell loss is minimal (85% recovery of input) and random, and cell viability, as measured by trypan blue staining, is high (greater than 95%). Factors interfering with separation are also described: a low loading flow rate, the presence of an excess of mature red cells, and the absence of fetal calf serum resulted in poor cell recovery and viability. The use of heparin as the anticoagulant resulted in cell loss, decrease in cell viability, and an ineffective separation. Separation of cells from normal bone marrow by centrifugal elutriation should facilitate the study of bone marrow cell interactions and the purification of individual types of bone marrow cells.

The inhibitory effect of various prostaglandin analogues on the anchorage independent growth of murine and human melanoma cells was measured. PGA analogues (which were modified at C-16 and C-18) did not demonstrate any major improvement in activity over PGA alone. These included 16,16-dimethyl PGA1, 16,16-dimethyl-PGA2, 16,16-dimethyl-18-oxa-PGA2 and trans-delta-2-15-alpha acetoxy-16,16-dimethyl-18-oxa-11-deoxy-PGE1-methylester. The thromboxane synthetase inhibitor, U51605, demonstrated weak anti-proliferative activity. PGD2 (with a ketone at C-11 versus C-9 for PGA and PGE) was the most potent prostaglandin tested. Cells from melanoma lines displayed species differences in their sensitivities. PGA1 and PGE1 were the most potent inhibitors of the anchorage independent growth of murine melanoma cells. On human melanoma cells PGD2 was the most active prostaglandin, 2-3 times more potent than PGA1; PGE1 was a very weak inhibitor.

We measured the effect of 6 standard (Adriamycin, BCNU, DTIC, melphalan, vinblastine, actinomycin D) and 3 Phase II agents (cis-platinum, vindesine, AMSA) on melanoma colony-forming units (CFU) in soft agar from biopsies of 50 patients with metastatic melanoma. Melanoma CFU demonstrated marked heterogeneity in chemosensitivity to these 9 drugs. Reduction in survival of CFU below 38% at one-tenth the pharmacologically achievable 1h concentration (our operational definition of chemosensitivity) was obtained in only 19% of 200 in vitro trials, and was usually the same whether or not patients had been exposed to prior chemotherapy, suggesting that melanoma CFU are inherently resistant to presently available chemotherapeutic drugs. The soft-agar assay was 86% accurate (25/29 cases) in identifying drugs to which the tumour was resistant in vivo, and 63% accurate (12/19 trials) in identifying drugs to which the tumour was clinically sensitive, counting mixed responses as responses. In contrast, if mixed responses were classified as progressive disease, the accuracy of identification of sensitivity fell to 42% (8/19 trials). These investigations furnish a quantitative description of the chemosensitivity of human metastatic melanoma CFU. Additionally, these studies serve as a useful step towards the development of an in vitro chemosensitivity test for human melanoma, and provide an operational quantitative basis for further exploration of in vitro-directed therapy in metastatic neoplasms.

Laryngeal papillomatosis often involves a relentless growth of papillomas on the vocal cords, requiring repeated excisions to maintain an adequate airway. Because of its antiproliferative effects on epithelial tissues, 13-cis-retinoic acid (0.5-2.0 mg/kd/day p.o.) was used in five patients whose disease was poorly controlled by laser beam surgery. Control of disease for 24+, 5+, and 12 months has been achieved in three of the patients, with two complete and one partial responses. Side effects of treatment were mild and rapidly reversible, following a 25-50% reduction in drug dose.

Terminal deoxynucleotidyl transferase activity was studied in 42 whole human thymuses of different ages (2 days to 70 years). The results indicate that enzyme activity is high perinatally, falls soon after birth, rises to maximal activity early in life, and falls thereafter. Enzyme activity in homologous thymocytes of 9 thymuses ranging from 2 days to 105 months was also determined. The relative enzyme activity (thymocyte/thymus) did not remain constant with age. These results suggest that age-related changes in thymus terminal transferase activity occur which do not reflect a simple coordinate regulation of thymocyte and non-thymocyte elements.

Pimozide, a potent neuroleptic which inhibits the release of pituitary releasing factors and is an effective dopamine antagonist, was administered to 30 patients with previously treated metastatic melanoma. Six patients were inevaluable because of poor drug tolerance (two), disease progression within 1 week and death within 2 weeks (three), and death from other causes (one). Among the 24 evaluable patients, two had complete response, two had partial response, and two had disease stabilization. Responses were observed in soft tissue, lymph nodes, liver, and lung. Toxic effects consisted of extrapyramidal manifestations in nine patients and malaise in seven. Pimozide has activity in patients with previously treated metastatic melanoma (17% response rate in evaluable patients) and merits consideration of further study in combination regimens.

A phase I trial was conducted of the vitamin A derivative beta-all-trans-retinoic acid (vitamin A acid; TRA), delivered via a collagen sponge and cervical cap for mild or moderate intraepithelial cervical neoplasia. On the basis of known skin and mucosal membrane toxicity, a concentration of 0.05% TRA in a cream-based vehicle was selected as the starting dose and was escalated later with the use of a modified Fibonacchi scale. The delivery device and the TRA were changed daily for 4 days, and side effects were assessed on days 1, 2, 3, 4, 8, and 30 by clinical and colposcopic examination. Vaginal, cervical, and systemic toxicity were evaluated in 35 patients. No dose-related systemic effects were found; mild cervical inflammation increased in many patients at higher doses. Unacceptably high vaginal toxicity was reached at a TRA concentration of 0.484%. A concentration of 0.372% TRA is recommended for use in phase II trials in mild and moderate cervical intraepithelial neoplasia.

Three short-term human melanoma cell lines were tested for sensitivity to human recombinant alpha-tumor necrosis factor (TNF) in a semisolid agar colony formation assay. Cells from three pigmented and one amelanotic strain displayed low sensitivity to TNF. The ID50 for the inhibition of melanoma colony formation ranged from 2,500 to 20,000 U/ml. We then tested the ability of human recombinant alpha-interferon (IFN-alpha) and gamma-interferon (IFN-gamma) to interact with TNF to inhibit melanoma colony formation. Analysis of the TNF-IFN mixtures using the median effect method demonstrated that both IFNs interacted synergistically with TNF to inhibit melanoma colony formation. On a unit basis, IFN-gamma was more active with TNF than IFN-alpha. The addition of the second interferon to the mixture enhanced the ability of TNF to promote the cytolysis of human melanoma cells. The enhanced killing effect seen with the combination of IFN-alpha, IFN-gamma, and TNF suggests an interesting strategy for the treatment of human melanoma.

Dysplasia of the uterine cervix is a recognized preneoplastic condition. Because of the observed ability of retinoids to reverse other dysplastic conditions in vitro and in vivo, a number of clinical studies have been carried out of the effect of these agents on cervical dysplasia, with the object of developing a means of chemoprevention of cervical malignancies in women at risk. We have conducted phase I and II trials of topical tretinoin (retinoic acid and Retin-A) delivered by means of a cervical cap and inert collagen sponge system. The results of these studies warranted a phase III trial, which is now underway. The outcome of the latter investigation will have important implications, not only for the management of patients with cervical dysplasia but also for therapeutic approaches to other precancerous conditions.

Accurate descriptions of the kinetics of cell growth in semi-solid agar clonogenic systems have been difficult because the number of cells in colonies of different sizes is largely unknown. We stained and removed tumor cell colonies from agar, directly counted their cells, and established equations to quantitate the number of cells within colonies of different sizes. We used these equations to quantitate, in terms of cell number and volume, the total amount and kinetics of clonogenic cell proliferation from biopsies of human melanoma and cell lines of several different tumor types. Daily observations of cells in agar and serial photography indicated a 0- to 4-day delay in the onset of proliferation in agar followed by rapid growth and then abrupt cessation of proliferation. We quantified the extent of proliferation of cells from melanoma biopsies of seven patients and 11 cell lines after they were allowed to proliferate in agar until they stopped. Approximately 10% of cells divided one to five times while only 0.01% divided six to nine times. The total number of cells within the colonies at the end of growth was different while the total volume of cells within the colonies per plate was similar; approximately 10(9) microns 3 cellular volume per plate represents an upper limit for proliferation within the closed, nonrefed bilayer agar system. Previous replating studies using the same biopsy cells have shown that clonogenic melanoma cells can self-renew and have more proliferative capacity than that expressed during primary colony formation. Thus, the clonogenic assay only measured initial proliferative capacities. Furthermore, variable delays in the onset of proliferation may contribute to the heterogeneity of colony size within clonogenic assays.

Four patients with refractory cutaneous T-cell lymphoma (mycosis fungoides) were treated with 13-cis-retinoic acid. Near complete clearing of extensive tumours and plaques was seen in one patient, who remains in partial remission with continued improvement after fifteen months. Two patients showed improvement in pruritus and 50% reduction in plaques by four and six weeks, respectively. The fourth patient had improvement in pruritus and clearing of plaques, but dryness and scaling necessitated reduction and eventually withdrawal of the treatment.

Of 48 cases of phenothiazine poisoning that were analyzed, 34 were attributed to suicide attempts, nine to accidental ingestion, and five to drug reactions. As outpatient treatment of schizophrenia increases, cases of over-dose with phenothiazine drugs may be expected to increase also. The prescribing of multiple phenothiazines and antidepressants is probably contributory to the occurrence of mixed drug ingestions. The symptoms and signs of phenothiazine poisoning are largely predictable if the atropine-like, alpha-blocking, quinidine-like, and extrapyramidal actions of phenothiazines are appreciated. Unexplainable tachypnea and paradoxical miosis were noted in severe cases. In one case in the study phenothiazine intoxication was present in the newborn infant of a schizophrenic mother.