Cutaneous malignant melanoma: A study of skin cancer in the United States from 1973--1997

Cutaneous malignant melanoma (CMM) is the cancer of the melanocytes, the cells that produce the pigment melanin, and is an aggressive skin cancer that is most prevalent in the white population. Although most cases of malignant melanoma are white, black and other non-white populations also develop this disease. However, the etiologic factors involved in the development of melanoma in these lower-risk populations are not well known. Generally, survival rates of malignant melanoma have been found to be lower in blacks than for whites with similar stage of disease at diagnosis. ^ This study presents an analysis of the differences in survival between black and white cases with malignant melanoma of the skin as the only or first primary cancer, found in the National Cancer Institute Surveillance, Epidemiology and End Results (SEER) cancer registry from 1973 to 1997. A total of 54,193 cases of CMM were diagnosed in black and white patients between 1973 and 1997. Black patients tended to be older, with a mean age of 64.46 years, compared to 53.14 years for white patients. Eighty-nine percent of patients were diagnosed with CMM as the only cancer. (Abstract shortened by UMI.)^

Investigation of the mechanism of increased melanoma incidence in UV -irradiated mouse skin

The availability of transplantable, syngeneic murine melanomas made it possible to study the potential effects of UV radiation on the growth and progression of melanomas in an animal model. The purpose of my study was to determine how UV-irradiation increases the incidence of melanoma out-growth, when syngeneic melanoma cells are transplanted into a UV-irradiated site. Short term intermittent UVB exposure produces a transitory change in the mice which allows the increased outgrowth of melanoma cells injected into the UV-irradiated site. One possible mechanism is an immunomodulatory effect of UVR on the host. An alternative mechanism to account for the increased tumor incidence in the UV-irradiated site, is the release of inflammatory mediators from UV-irradiated epidermal cells. A third possibility is that UVR could induce the production and/or release of melanoma-specific growth factors resulting in increased melanoma outgrowth.^ My first step in distinguishing among these different possible mechanisms was to characterize further the conditions leading to increased development of melanoma cells in UV-irradiated mouse skin. Next, I attempted to determine which of the 3 proposed mechanisms was most likely. To do this, I defined the specificity of the effect by examining the growth of additional C3H tumorigenic cell lines in UV-irradiated skin. Second, I determined the immunogenicity of these tumor cell lines. The tumor cell lines exhibiting increased tumor incidence are restricted to those tumor cell lines which are immunogenic in normal C3H mice. Third, I determined the effect of UVR on melanoma development did not occur in immunosuppressed mice.^ Because of results from these three lines of investigation suggested that the effect was immunologically mediated, I then investigated whether specific immune reactions were affected by local UV irradiation. To accomplish this, I investigated the effect of UVR on cutaneous immune cells and on induction of contact hypersensitivity (CHS), and I also determined the effect of UVR on the development and the expression of systemic immunity against the melanoma cells. There is no clear cut relationship between the number of Langerhans or Thy1+ cells and the UV effect on tumor incidence. Furthermore, there was no suppression of CHS in the UV-irradiated mice. While the development of systemic immunity is significantly reduced, it appears to be sufficient to provide in vivo immunity to tumor challenge. However the elicitation of tumor immunity in immunized mice can be abrogated if tumor challenge occurs in the site of UV irradiation. This investigation provides new information on an effect of UVR on the elicitation of tumor immunity. Furthermore, it indicates that UV radiation can play a role in the development of melanoma other than just in the transformation of melanocytes. ^

Regulation of apoptosis during the pathogenesis of non -melanoma skin cancer

Previous studies from our lab have shown distinctive patterns of expression of bcl-2 gene family members in human nonmelanoma skin cancer (NMSC). To further evaluate the significance of these observations and to study the effects of cell death deregulation during skin carcinogenesis, we generated a transgenic mouse model (HK1.bcl-2) using the human keratin 1 promoter to target the expression of a human bcl-2 minigene to the epidermis. Transgenic protein expression was confirmed in all the layers of the epidermis except the stratum corneum using immunohistochemistry. Multifocal epidermal hyperplasia, without associated hyperkeratosis, was observed in newborn HK1.bcl-2 mice. Immunofluorescence staining using monoclonal antibodies specific for a variety of differentiation markers revealed aberrant expression of keratin 6 (K6) in the transgenic epidermis. Epidermal proliferative indexes, assessed by anti-BrdUrd immunofluorescence staining, were similar in control and transgenic newborn mice, but suprabasal proliferating cells were seen within the hyperplastic areas of the transgenic mouse skin. Spontaneous apoptotic indices of the epidermis were similar in both control and HK1.bcl-2 transgenic newborn mice, however, after UV-B irradiation, the number of "sunburn cells" was significantly higher in the control compared to the HK1.bcl-2 transgenic animals.^ Adult HK1.bcl-2 and control littermate mice were used in UV-B and chemical carcinogenesis protocols including DMBA + TPA. UV-B irradiated control and HK1.bcl-2 mice had comparable incidence of tumors than the controls, but the mean latency period was significantly shorter in the HK1.bcl-2 transgenic. Both control and transgenic animals included in chemical carcinogenesis protocols required application of both the initiating (DMBA) and promoting (TPA) agents to develop tumors. The frequency, number, and latency of tumor formation was similar in both groups of animals, however, HK1.bcl-2 mice exhibited a rate of conversion from benign papilloma to carcinoma 2.5 times greater than controls.^ Similar carcinogenesis experiments were performed using newborn mice. HK1.bcl-2 mice treated with UV-B plus TPA have a three fold greater incidence of tumor formation compared to controls littermates. HK1.bcl-2 transgenic animals also exhibited a shorter latency for papilloma formation when treated with DMBA plus TPA.^ HK1.bcl-2/v-Ha-ras double transgenic mice shared phenotypic features of both HK1.v-Ha-ras and HK1.bcl-2 transgenic mice, and exhibited focal areas of augmented hyperplasia. These double transgenic mice were susceptible to tumor formation by treatment with TPA alone.^ Cultures of primary keratinocytes were established from control, HK1.bcl-2, HK1.Ha-ras, and HK1.bcl-2/v-Ha-ras newborn mice. Cell viability was determined after exposure of the cells to UV-B irradiation, DMBA, TPA, or TGF-$\beta$1. Internucleosomal DNA fragmentation ("ladders") and morphological cellular changes compatible with apoptotic cell death were observed after the application of all these agents. HK1.bcl-2 keratinocytes were resistant to cell death induction by all of these agents except TGF-$\beta$1. HK1.Ha-ras cells had a higher spontaneous rate of cell death which could be compensated by co-expression of bcl-2.^ These findings suggest that bcl-2 dependent cell death suppression may be an important component of multistep skin carcinogenesis. ^

Participation of bcl -2 and bax in epidermal homeostasis and non-melanoma skin cancer

Non-melanoma skin cancer (NMSC) is the most frequently diagnosed form of cancer in United States. As in many other cancers, this slow growing malignancy manifests deregulated expression of apoptosis regulating proteins including bcl-2 family member proteins. To understand the role of apoptosis regulating protein in epidermal homeostasis and progression of NMSC, we investigated keratinocyte proliferation, differentiation and tumorigenesis in bcl-2 and bax null mice. The rate and the pattern of proliferation and spontaneous cell death were the same between the null and the control mice. Both bcl-2 and bax null epidermis showed decreased levels of cytokeratin 14 expression compared to the control littermates. Also, the gene knock out mice showed higher expression of cytokeratin 1 and loricrin in epidermis compared to the control mice. The apoptotic response to genotoxic agent, UV radiation (UVR), was assessed by counting sunburn cells. The bax null keratinocytes showed a resistance to apoptosis while bcl-2 null mice showed an increased susceptibility to cell death compared to the control mice. Moreover, we demonstrated an increase in tumor incidence in bax null mice compared to control littermates in the in vivo chemical carcinogenesis study. Next, we examined the tumor suppressor role of bax protein in NMSC by studying its participation in repair of UVR-mediated DNA lesions. In UVR treated primary keratinocytes from bax deficient mice, the level of CPD remaining was twice that of control cells at 48 hours. Similar results were obtained using embryonic fibroblasts from bax null and bax +/+ embryos, and also with a bax deficient prostate cancer cell line in which bax expression had been restored. However, the repair rate of 6-4 PP was unaffected by the absence of bax protein in all three of above mentioned cell types. In conclusion, bax protein may have a dual function in its role as tumor suppressor in NMSC. Bax may directly or indirectly facilitate DNA repair, or programmed cell death if DNA damage is too severe, thus, in either function, preserving genomic integrity following a genotoxic event. ^

The role of endogenous IFN-beta in cutaneous melanoma: Consequences of epidermal hyperplasia

The purpose of this study was to characterize epidermal hyperplasia overlying malignant melanoma, to determine the mitogenic factor responsible for the induction of this hyperplasia and to investigate its biological consequence. Whether increased keratinocyte proliferation overlying melanoma is due to production of growth factors by the tumor cells or to other mechanisms is unknown. Epidermal hyperplasia overlying human melanoma was found overlying thick (>4.0mm), but not thin (<1.0mm) tumors. Immunostaining of the sections for growth factors related to angiogenesis revealed that epidermal hyperplasia was associated with loss of IFN-β production by the keratinocytes directly overlying the tumors. Since previous studies from our laboratory have demonstrated that exogenous administration of IFN-β negatively regulates angiogenesis, we hypothesize that tumors are able to produce growth factors which stimulate the proliferation of cells in the surrounding tissues. This hyperplasia leads to a decrease in the endogenous negative regulator of angiogenesis, IFN-β. ^ The human melanoma cell line, DM-4 and several of its clones were studied to identify the mitogenic factor for keratinocytes. The expression of TGF-α directly correlated with epidermal hyperplasia in the DM-4 clones. A375SM, a human melanoma cell line that produces high levels of TGF-α, was transfected with a plasmid encoding full-length antisense TGF-α. The parental and transfected cells were implanted intradermally into nude mice. The extent of epidermal hyperplasia directly correlated with expression of TGF-α and decreased production of IFN-β, hence, increased angiogenesis. ^ In the next set of experiments, we determined the role of IFN-β on angiogenesis, tumor growth and metastasis of skin tumors. Transgenic mice containing a functional mutation in the receptor for IFN α/β were obtained. A375SM melanoma cells were implanted both s.c. and i.v. into IFN α/βR −/− mice. Tumors in the IFN α/β R −/− mice exhibited increased angiogenesis and metastasis. IFN α/βR −/− mice were exposed to chronic UV irradiation. Autochthonous tumors developed earlier in the transgenic mice than the wild-type mice. ^ Collectively, the data show that TGF-α produced by tumor cells induces proliferation of keratinocytes, leading to epidermal hyperplasia overlying malignant melanoma associated with loss of IFN-β and enhanced angiogenesis, tumorigenicity and metastasis. ^

NRAS and BRAF mutations in primary cutaneous melanoma: A comparison of mutation rates between radial and vertical growth phases in individual tumors

Primary cutaneous melanoma is a cancer arising from melanocytes in the skin. In recent decades the incidence of this malignancy has increased significantly. Mortality rates are high for patients with tumors measuring over a few millimeters in thickness. Response rates to conventional radiation and chemotherapy are very low in patients with metastatic melanoma. New therapies targeting melanoma’s aberrant cell signaling pathways such as the MAP Kinase pathway are being developed. Mutations of NRAS and BRAF genes are quite common in cutaneous melanoma and lead to constitutive activation of the MAP Kinase pathway. This study tests the hypothesis that NRAS and BRAF mutations increase as a tumor progresses from the noninvasive radial growth phase (RGP) to the invasive vertical growth phase (VGP). Laser capture microdissection was used to obtain separate, pure tumor DNA samples from the RGP and VGP of thirty primary cutaneous melanomas. PCR was used to amplify NRAS exon 2 and BRAF exon 15 tumor DNA. The amplified DNA was sequenced and analyzed for mutations. An overall mutation rate of 74% was obtained for the twenty-three melanomas in which there were complete sequence results. With the exception of one melanoma NRAS and BRAF mutations were mutually exclusive. All seven NRAS exon 2 mutations involved codon 61. Three of these melanomas had mutations in both the RGP and VGP. The remaining four tumors were wild type for NRAS exon 2 in the RGP but mutated in the VGP. Of the fifteen BRAF exon 15 mutated melanomas all but one involved codon 600. Twelve of the fifteen BRAF exon 15 mutations were the T1799A type. Nine of the fifteen BRAF mutated tumors had the same mutation in both the RGP and VGP. Five of fifteen melanomas had wild type RGP DNA and BRAF exon 15 mutated VGP DNA. A single melanoma had BRAF exon 15 mutated DNA in the RGP and wild type DNA in the VGP. Overall, these results suggest a trend toward the acquisition of NRAS and BRAF mutations as cutaneous melanomas change from a noninvasive to an invasive, potentially deadly cancer.^

Psychosocial measures reported by parents in studies of skin cancer prevention

Background. Because it is important to minimize children's sun exposure to reduce skin cancer risk, much of the extensive skin cancer prevention literature consists of studies of children's sun protection, sun avoidance and ultraviolet radiation (UVR) exposure. Little attention has been focused on the measurement of psychosocial constructs in these studies. Identification of the psychosocial correlates or determinants of children's skin cancer risk or risk-reduction behavior is critical to more fully understand and predict behavior. Furthermore, psychosocial variables may be influenced by interventions to reduce risk. Thus, it is important to examine the psychosocial measures used in studies of children's skin cancer prevention. Information on the validity and reliability of psychosocial measures may increase confidence in study findings based on these measures. In particular, self-efficacy and barriers are key constructs in several major theoretical frameworks and parental measures have been associated with children's sun protection. However, there is conceptual overlap of self-efficacy and barriers measures and little is known about the psychometric properties of these measures.^ Study Aims and Methods. The overall goal of this dissertation was to examine the measurement of psychosocial constructs relevant to children's skin cancer prevention. Because children depend primarily on their parents for skin cancer prevention, measures of parents' psychosocial constructs are the focus. Study 1 was a systematic review of parental psychosocial measures used in studies of children's sun protection, sun avoidance and UVR exposure. The specific aims of Study 1 were to (1) describe psychosocial measures reported by parents, including available information on the psychometric properties of these measures and their use in analyses and (2) provide recommendations for the development, refinement and standardized reporting of measures. ^ Study 2 examined the psychometric properties of measures of parental self-efficacy and barriers regarding children's sun protection. Melanoma patients (N=205) who were parents of children ≤ 12 years of age completed a telephone interview that included self-efficacy and barriers measures specific to sunscreen, clothing, shade and limiting time outdoors. The specific aims of Study 2 were to (1) use a confirmatory factor analytic approach to examine the factorial validity of parental self-efficacy and barriers measures, (2) examine the convergent and discriminant validity of behavior-specific measures of self-efficacy and barriers and (3) assess the reliability of item and scale measures.^ Results. In Study 1, a search of standard databases yielded 48 eligible studies. Most studies assessed only one or two psychosocial constructs. Knowledge was measured most frequently. There was little discussion of measure source, development, theoretical background or psychometric properties, besides internal consistency reliability. There was conceptual overlap of some measures. In Study 2, confirmatory factor analytic findings supported the factorial validity of the self-efficacy and barriers measures. When all eight self-efficacy and barriers measures were included in the same model, a modified eight-factor model adequately fit the data, providing preliminary evidence that the measures are distinct. Measure associations supported the convergent validity of all measures and the discriminant validity of most measures. The self-efficacy and barriers measures were reliable.^ Conclusions. Recommendations based on the literature review include developing and refining psychosocial measures based on theory. Describing a measure's theoretical basis and psychometric properties would facilitate critical evaluation. Standardized reporting of source, development, theory, construct, items and analytic role would facilitate comparison of findings, continual refinement and future applications of measures. In the validation study, self-efficacy and barriers measures were examined in a sample of parents with a personal history of melanoma. Findings suggested that these measures are valid and reliable for use in studies of children's sun protection. There was preliminary evidence that these measures are distinct but additional study is needed. ^

CHARACTERIZATION OF DIFFERENTIATION AND PROGNOSTIC BIOMARKERS ON CD8+ TUMOR-INFILTRATING LYMPHOCYTES IN METASTATIC MELANOMA

CD8+ cytotoxic T lymphocytes (CTL) frequently infiltrate tumors, yet most melanoma patients fail to undergo tumor regression. We studied the differentiation of the CD8+ tumor-infiltrating lymphocytes (TIL) from 44 metastatic melanoma patients using known T-cell differentiation markers. We also compared CD8+ TIL against the T cells from matched melanoma patients’ peripheral blood. We discovered a novel subset of CD8+ TIL co-expressing early-differentiation markers, CD27, CD28, and a late/senescent CTL differentiation marker, CD57. This CD8+CD57+ TIL expressed a cytolytic enzyme, granzyme B (GB), yet did not express another cytolytic pore-forming molecule, perforin (Perf). In contrast, the CD8+CD57+ T cells in the periphery were CD27-CD28-, and GBHi and PerfHi. We found this TIL subset was not senescent and could be induced to proliferate and differentiate into CD27-CD57+, perforinHi, mature CTL. This further differentiation was arrested by TGF-β1, an immunosuppressive cytokine known to be produced by many different kinds of tumors. Therefore, we have identified a novel subset of incompletely differentiated CD8+ TIL that resembled those found in patients with uncontrolled chronic viral infections. In a related study, we explored prognostic biomarkers in metastatic melanoma patients treated in a Phase II Adoptive Cell Therapy (ACT) trial, in which autologous TIL were expanded ex vivo with IL-2 and infused into lymphodepleted patients. We unexpectedly found a significant positive clinical association with the infused CD8+ TIL expressing B- and T- lymphocyte attentuator (BTLA), an inhibitory T-cell receptor. We found that CD8+BTLA+ TIL had a superior proliferative response to IL-2, and were more capable of autocrine IL-2 production in response to TCR stimulation compared to the CD8+BTLA- TIL. The CD8+BTLA+ TIL were less differentiated and resembled the incompletely differentiated CD8+ TIL described above. In contrast, CD8+BTLA- TIL were poorly proliferative, expressed CD45RA and killer-cell immunoglobulin-like receptors (KIRs), and exhibited a gene expression signature of T cell deletion. Surprisingly, ligation of BTLA by its cognate receptor, HVEM, enhanced the survival of CD8+BTLA+ TIL by activating Akt/PKB. Our studies provide a comprehensive characterization of CD8+ TIL differentiation in melanoma, and revealed BTLA as a novel T-cell differentiation marker along with its role in promoting T cell survival.