Plantar lentiginous melanoma. A clinicopathologic study.

Twenty cases of plantar melanoma treated at Ellis Fischel State Cancer Center (EFSCC) since 1940 are analyzed to determine the clinical and histologic features of prognostic significance. The EFSCC experience confirms and extends the observations made in other recent studies of plantar melanoma. This clinical pathologic study suggests that one of the histologic patterns, the lentiginous growth pattern, shares some biologic features with lentigo maligna, namely the relatively indolent growth phase. Plantar melanomas may remain latent for a number of years making these patients good candidates for therapeutic cures if detected early.

Evaluation of premalignant and malignant lesions during the induction of mouse melanomas.

The objective of the present study was to evaluate the malignant and premalignant lesions that arise in C57BL/6 mice after treatment with 7,12-dimethylbenz(a)anthracene and croton oil. Tissues from 70 treated mice were evaluated by histological and transplantation techniques, and 17 (24%) were found to have malignant tumors. Eleven of the tumors were diagnosed as malignant melanomas, three as spindle cell sarcomas, and three as squamous cell carcinomas. The incidence of malignant melanomas (15.7%) in this group of mice was similar to that in our initial study on the induction of melanomas with 7,12-dimethylbenz(a)-anthracene and croton oil, in which two of 20 mice developed malignant melanomas. Mice that developed melanomas had been treated with croton oil for an average of 7 mo, and the mean latent period for tumor development was 11 mo. Seven of eight melanomas grew rapidly after transplantation to syngeneic C57BL/6 mice. Pigmented nevi and/or draining lymph nodes from nine of 11 mice with melanomas grew progressively after transplantation to athymic nude mice. Pooled nevi from one mouse with no apparent tumors grew into a histologically malignant melanoma after transplantation to a nude mouse. Nevi from three mice with sarcomas, one mouse with a carcinoma, and 42 tumor-free treated mice failed to grow in nude transplant recipients. Thus, only nevi from mice with either apparent or occult malignant melanomas exhibited progressive growth in nude mice. These results confirm that malignant melanomas can be induced in C57BL/6 mice at a regular, predictable rate and further indicate that this is an excellent system in which to study melanoma induction, progression, and therapy.

Phenotypic instability of mouse melanomas after propagation in vivo and in vitro.

This study was designed to evaluate the phenotypic stability of the carcinogen-induced JB/MS and JB/RH melanomas. The JB/MS melanoma maintained its original slow-growing melanotic phenotype during in vivo passage over a 2-yr period. However, both melanin production and tumorigenicity decreased rapidly after propagation of JB/MS cells in vitro. The JB/RH melanoma became essentially amelanotic after the second transplantation in vivo. Cultured JB/RH cells produced tumors identical to those obtained by transplantation of JB/RH tumor fragments. However, after propagation in vitro for 2 yr, the JB/RH cell line decreased in tumorigenicity, requiring 10 times as many cells to produce tumors in C57BL/6 mice as did the original cell line. The JB/RH melanoma was highly immunogenic in syngeneic C57BL/6 mice, and passage of JB/RH cells through immunized mice resulted in tumors that were significantly more tumorigenic in normal mice than were JB/RH cells that had been passed through either nude or sublethally irradiated mice. These results indicate that, in studies of primary mouse melanomas, it is essential to: (a) limit the number of tumor passages; (b) choose methods of propagation that will preserve the original phenotype; and (c) distinguish those properties produced by technical manipulation from those produced by true tumor progression.

Phase I clinical trial of an MAF-containing lymphoblastoid cell line supernatant.

A preliminary Phase I evaluation of macrophage activating factor (MAF) derived from the partially purified supernatant of human RPMI-1788 lymphoblastoid cell line was performed in 4 parts in 39 patients with advanced cancer. The first two parts used subcutaneous routes of administration and the second two parts used a 4-h intravenous infusion method. Individual doses ranged from 0.1 ml (1.7 mg protein) to 100 ml (1,700 mg protein). Subcutaneous dose was limited by the volume of administered material, and an attempt to use a concentrate of the supernatant resulted in severe local skin reactions. Larger doses given intravenously were well tolerated. Resultant toxicity was mild and consisted of transient fever and chills. One patient with malignant melanoma had a complete response of a 3-cm skin metastasis; one patient with breast cancer had disappearance of a skin nodule while visceral disease progressed; and one patient with histiocytic lymphoma had resolution of a conjunctival lesion. Treatment in many patients was associated with an increase in absolute peripheral lymphocytes. In the high-dose intravenous group, a statistically significant increase in the phagocytic index of peripheral blood leukocytes was noted. Lymphoblastoid MAF appears to be relatively safe to administer and has promise both as an antitumor agent and in the treatment of other altered immune conditions.

Ciliated adenocarcinoma of the endometrium.

Two cases of ciliated endometrial adenocarcinoma which came to attention in less than one year are described. This entity deserves emphasis, as its ciliation may distract from its histological malignant features. Association of cilia with atypical nuclear features in the same cell is the diagnostic characteristic which distinguishes it from non-ciliated adenocarcinoma overrunning non-malignant ciliated endometrium. The ultrastructural detail of the cilia of malignant cells does not differ from that of non-malignant endometrial glandular epithelium. Our cases raise the question of a possible association of estrogenic effect with ciliated endometrial adenocarcinoma.

Flow cytometric determination of estrogen receptors in intact cells.

Flow cytometry has been used to detect estrogen receptor (ER) in intact cells, using 1-(N)- fluoresceinyl estrone thiosemicarbazone ( 17FE ), a ligand shown to have sufficient affinity and specificity to identify high-affinity receptors. For each concentration of 17FE , two fluorescent distributions were obtained: (a) "total" fluorescence due to 17FE alone; and (b) diethylstilbestrol-inhibited fluorescence ("nonspecific"). The mean fluorescence intensity for both the "total" (MT) and "nonspecific" (MN) distributions was calculated at each ligand concentration by obtaining the total brightness of each sample, normalized to the number of cells analyzed. A specific binding curve was constructed over a broad range of 17FE concentrations (1.5 X 10(-9) to7 .5 X 10(-7) M) by plotting the mean specific fluorescence intensity (MS = MT-MN) at each 17FE concentration. Saturable binding was demonstrated for a known ER-positive cell line (MCF-7) at low ligand concentrations (5 to 10 X 10(-8) M), while no specific binding was obtained for a known ER-negative cell line (MDA-231). The Kd was estimated from the specific binding curve of MCF-7 cells as the concentration of 17FE at half-maximal binding. When corrected for the binding activity relative to estradiol, a Kd of 18 X 10(-10) M was obtained. The flow cytometer-generated distributions give a qualitative estimate of the proportions of ER-rich and ER-poor cells. Quantitation of ER heterogeneity will require a mathematical algorithm expressing the difference between the "total" and "nonspecific" distributions. These studies demonstrate that flow cytometry and an appropriate ligand can detect and partially describe ER heterogeneity in intact cells.

Cell-mediated immune reactivity of Sinclair melanoma-bearing swine to 3 M KCl extracts of swine and human melanoma.

Leukocyte migration inhibition (LMI) assays were performed using 3 M KCI extracts to detect cell-mediated immune reactions against tumor-associated antigens (TAA) of swine melanoma. Positive LMI reactivity was 70% or greater in melanoma-bearing swine compared to 10% reactivity or less in normal swine tested with extracts of either fresh or tissue-cultured swine melanoma cells. Positive LMI reactivity was 10% or less in both melanoma and normal swine tested with extracts of normal fetal and adult swine tissues. Extracts of tissue-cultured swine melanoma and human melanoma cells were equally capable of stimulating positive LMI reactivity in melanoma swine and human melanoma patients; normal swine and human donors demonstrated 10% or less positive LMI reactivity to these extracts. Positive LMI reactivity was not stimulated in swine melanoma or human melanoma donors by extracts of tissue-cultured human breast tumor cells or fresh and tissue-cultured mouse B16 melanoma cells. Evaluation of these results indicate that melanoma-bearing swine develop cell-mediated immune reactivity toward TAA on swine melanomas and, inasmuch as LMI assays were performed with allogeneic extracts, suggests the presence of common TAA on swine melanomas. Furthermore, these data suggest that swine and human melanoma cells may share some common TAA and that this common TAA is not expressed on mouse B16 melanoma cells.

Growth and regression of cutaneous melanomas in Sinclair miniature swine.

Previous studies have demonstrated pathologic similarities between human melanoma and the spontaneous melanoma in the Sinclair miniature swine (SMS) with respect to cutaneous histologic features and patterns of metastasis. The current biopsy series, correlating growth curves and histopathologic features of cutaneous melanomas, was undertaken for documentation of the histologic events associated with the successful regression of melanoma in the SMS. Cutaneous growth and regression was characterized by a series of cellular events that eventually led to depigmentation and scar formation. Mononuclear inflammatory infiltrates, seen in over half of the 104 biopsies, showed several temporal and topographic distribution patterns, similar to that described in human melanoma. Histopathologic observations in the SMS confirm clinical observations that the host can, with consistent effectiveness, react with the tumors to modify their biologic aggressiveness. Although regression is associated with lymphocytic and macrophage infiltration, the exact role of the immune response in the regression of the cutaneous melanoma remains to be elucidated.

Development of a new melanoma model in C57BL/6 mice.

In the present study, we induced melanomas in C57BL/6 mice by a single application of 7,12-dimethylbenz(a)anthracene to the scapular region of 4-day-old mice, followed by twice-weekly applications of croton oil. Of 20 mice treated, melanomas arose in two female littermates. The first melanoma (JB/MS) arose 16 weeks after initiation of treatment, and the second melanoma (JB/RH) arose 23 weeks later. The melanomas maintained their melanotic appearance after s.c. transplantation to normal C57BL/6 mice and metastasized spontaneously in the transplant recipients. To our knowledge, these are the first melanomas to have been induced in C57BL/6 mice since the B16 melanoma arose spontaneously in 1954. We feel that the JB/MS and JB/RH melanomas provide an excellent comparative system for studies done with the B16 melanoma. These melanomas of recent origin will also facilitate the investigation of biological, immunological, and biochemical parameters that influence the growth and metastasis of malignant melanomas.

Growth and spontaneous regression of swine melanoma: relationship of in vitro leukocyte reactivity.

A visual microcytotoxicity technique was used to evaluate the leukocyte reactivity of melanoma swine against allogeneic swine melanoma target cells. Peripheral blood leukocytes, which had been collected and cryopreserved in liquid nitrogen at various times during in vivo tumor growth and regression, were thawed and tested on the same day. Comparison of longitudinal leukocyte reactivity with in vivo tumor volume indicated that swine with regressing melanomas exhibited increased leukocyte reactivity during tumor regression. Swine with maximum tumor volumes less than 30,000 mm3 exhibited patterns of leukocyte reactivity that paralleled the patterns of in vivo tumor growth and regression. However, swine with maximum tumor growth and regression. However, swine with maximum tumor volumes greater than 30,000 mm3 demonstrated increased in vitro leukocyte reactivity at the time of maximum in vivo tumor volume. Histopathologic analyses revealed that increases in tumor volume were frequently a result of host inflammatory cells, particularly pigment-laden macrophages, infiltrating the tumors. Thus at the time of maximum tumor volume, malignant melanocytes were proportionately decreasing in number while pigment-laden macrophages were proportionately increasing. These studies provide additional evidence that the spontaneous tumor cell regression of swine melanoma is associated with immunologic events and that assays of leukocyte reactivity are useful in vitro correlates of host antitumor immunity.

Tyrosinase activity and isoenzyme distribution corresponding to growth and regression of melanoma in Sinclair miniature swine.

Malignant melanomas have been shown to contain high levels of monophenol monooxygenase (tyrosinase) enzyme activity; the enzyme is responsible for melanin synthesis. The melanoma of Sinclair miniature swine has a high incidence of spontaneous regression and thus provides a unique system for analyzing changes in tyrosinase activity at various tumor stages. Three tumor stages (progressively growing tumors, partially regressed tumors, and fully regressed tumors) were analyzed for tyrosinase activity. The progressing tumors were 34-fold higher than were the partially regressed lesions and 400-fold higher than were the fully regressed tumors. Histologically, the decrease in enzyme activity correlated with a loss of tumor cells. Sequential biopsies of tumors during the course of tumor development showed a positive correspondence between tumor volume and tyrosinase activity for the early and late stages of tumor growth and regression. Electrophoretic separation of tyrosinase preparations revealed three major tyrosinase "isoenzymes" whose relative abundance fluctuated during developmental increases and decreases in enzyme activity.

Adaptation of Sinclair swine melanoma cells to long-term growth in vitro.

Sinclair swine melanoma usually regresses in vivo. In the present study, swine melanoma cells were adapted to long-term growth in culture. The morphology of cultured melanoma cells ranged from dendritic to cuboidal, similar to that described for human melanoma cells. Doubling times of the swine melanoma cells were also similar to those of human melanoma cells in vitro. 3,4-Dihydroxy-L-phenylalanine oxidase-positive cells were detected by light microscopy, and melanin and premelanosomes were detected by electron microscopy. Cell cultures could be propagated from progressing, partially regressed, and primary cutaneous lesions, as well as from visceral metastases. Thus, it appears that, under these cell culture conditions, Sinclair swine melanoma cells can be adapted to prolonged growth in vitro.

Malignant melanoma in the Sinclair miniature swine: an autopsy study of 60 cases.

Sinclair miniature swine spontaneously develop multiple cutaneous melanomas which have the ability to metastasize and regress. This study, based on 60 necropsies, documents the similarity of the pathology of the cutaneous malignant melanomas and the organ distribution of metastasis to human melanoma. The invasive cutaneous melanomas have an intraepidermal component analogous to human superficial spreading melanoma. The pathology of the spontaneous regression, characterized by a series of cellular events beginning with a mononuclear inflammatory infiltrate and leading to depigmentation and fibrosis, is likewise similar to cutaneous regression in human melanoma. Just as with human melanoma,metastasis was correlated with deeply invasive cutaneous tumors. Because of both the biologic and histologic similarity of this animal model to human melanoma, the Sinclair miniature swine should serve as an important resource in continuing the study of melanoma.

Lymphocyte-tumor cell interaction in patients with head and neck cancer.

Peripheral lymphocytes from 12 patients with squamous cell carcinoma of the lead and neck were incubated with autologous tumor explants. Four of the 12 patients demonstrated lymphocyte induced tumor cytotoxicity. These lymphocytes adhered to the tumor cells and deposited a radioactive label from their surface onto tumor cells. The deposition of this label was associated with tumor death. Tissue sections from those patients who demonstrated lymphocyte cytotoxicity showed a marked plasmacytic infiltration. This was in contrast to non-responders where only a desmoplastic tissue response was observed with few inflammatory cells.

Rheumatoid factor: a cause of fals positive histoplasmin latex agglutination.

Ten of thirteen patients with positive histolatex agglutination titers of 1:32 or greater had no evidence of acute histoplasmosis. Three of these false positives had rheumatoid arthritis. A fourth had a rising mycoplasma complement fixation titer, and the fifth had a high titer of cold agglutinins. All of these are associated with abnormal immunoglobulin M production. To evaluate the role of rheumatoid factor in producing false positive histolatex agglutination, the histolatex test was performed on sera from 32 patients having rheumatoid factor at a titer of 1:40 or greater. Four of these sera agglutinated the histoplasmin-coated latex particles at titers of 1:32 or greater. Review of clinical records suggests the this reactivity is nonspecific. It is our purpose to call attention to rheumatoic factor as a cause of false positive histolatex agglutination.