Risk of melanocytic nevi and nonmelanoma skin cancer in children after allogeneic hematopoietic stem cell transplantation.

There is a known increased risk of skin cancer in the adult population after hematopoietic stem cell transplantation (HSCT). However, late dermatologic effects that children may experience after HSCT have not been well described. The primary objective of this study was to characterize nevi and skin cancers affecting children after allogeneic HSCT. A cross-sectional cohort study of 85 pediatric HSCT recipients and 85 controls matched for age, sex and skin phototype was performed at a single institution. All participants underwent a full skin examination. Median age at study visit was 13.8 years in HSCT patients with median time post-HSCT of 3.6 years. HSCT patients had significantly more nevi than control patients (median (range): 44 (0-150) vs 11 (0-94), P<0.0001). HSCT patients also had significantly more nevi >5 mm in diameter and atypical nevi than controls. Factors associated with increased nevus count included malignant indication for HSCT, pretransplant chemotherapy, TBI exposure and myeloablative conditioning. A total of 16.5% of HSCT patients developed cancerous, precancerous lesions and/or lentigines. Our study suggests that pediatric HSCT recipients have an increased risk of benign and atypical melanocytic proliferations and nonmelanoma skin cancer that can manifest even during childhood.

Melanocyte photobiology, ultraviolet radiation and melanoma.

Of all the organs of the human body, the skin is most commonly affected by malignancy, and ultraviolet radiation has long been implicated as the primary mutagenic exposure leading to the development of many cutaneous cancers. However, as research in this field has continued, it has become clear that the effect of ultraviolet radiation on the skin is quite complex. Distinct cell types within the skin exhibit differing responses to ultraviolet radiation, and even within the same cell type, divergent effects may be observed; depending on the dose or wavelength of the radiation, or the maturational state of the affected cell, unique responses can be elicited. Melanocytes form a minor component of the outermost layer of skin, but they have an enormous impact on not only the appearance of the skin, but also the ability of the skin to withstand exposure to ultraviolet radiation. In addition, melanocytes give rise to melanoma, one of the most deadly types of skin cancer. Clearly, it is critical that we achieve a better understanding of the effect of ultraviolet radiation on melanocytes, and that we clarify its role in the oncogenesis of melanoma. Although the picture is far from complete, the mechanisms by which melanocytes respond to ultraviolet radiation are beginning to be elucidated, and, as these pathways emerge, they offer new targets for chemopreventive and chemotherapeutic intervention.

"Lineage addiction" in human cancer: lessons from integrated genomics.

Genome-era advances in the field of oncology endorse the notion that many tumors may prove vulnerable to targeted therapeutic avenues once their salient molecular alterations are elucidated. Accomplishing this requires both detailed genomic characterization and the ability to identify in situ the critical dependencies operant within individual tumors. To this end, DNA microarray platforms such as high-density single-nucleotide polymorphism (SNP) arrays enable large-scale cancer genome characterization, including copy number and loss-of-heterozygosity analyses at high resolution. Clustering analyses of SNP array data from a large collection of tumor samples and cell lines suggest that certain copy number alterations correlate strongly with the tissue of origin. Such lineage-restricted alterations may harbor novel cancer genes directing genesis or progression of tumors from distinct tissue types. We have explored this notion through combined analysis of genome-scale data sets from the NCI60 cancer cell line collection. Here, several melanoma cell lines clustered on the basis of increased dosage at a region of chromosome 3p containing the master melanocyte regulator MITF. Combined analysis of gene expression data and additional functional studies established MITF as an amplified oncogene in melanoma. MITF may therefore represent a nodal point within a critical lineage survival pathway operant in a subset of melanomas. These findings suggest that, like oncogene addiction, "lineage addiction" may represent a fundamental tumor survival mechanism with important therapeutic implications.

Modulation of microphthalmia-associated transcription factor gene expression alters skin pigmentation.

The microphthalmia-associated transcription factor is implicated in melanocyte development and in the regulation of melanogenesis. Microphthalmia-associated transcription factor is thought to bind to the M-box promoter elements of tyrosinase, tyrosinase-related protein-1 and dopachrome tautomerase/tyrosinase-related protein-2 and transactivate these genes, resulting in increased pigmentation. Using a luciferase reporter construct driven by the microphthalmia-associated transcription factor promoter, we identified agents that modulate microphthalmia-associated transcription factor promoter activity. Changes in endogenous microphthalmia-associated transcription factor expression levels upon treatment with these agents were confirmed using northern and western blots, and their pigmentary modulating activities were demonstrated. Ultraviolet B irradiation and traditional Chinese medicine-1, a natural extract used in traditional Chinese medicine, upregulated microphthalmia-associated transcription factor gene expression and enhanced tyrosinase activity in vitro. Dihydrolipoic acid, lipoic acid, and resveratrol reduced microphthalmia-associated transcription factor and tyrosinase promoter activities. These agents also inhibited the forskolin- and ultraviolet B-stimulated promoter activities of these genes and significantly reduced tyrosinase activity in melanocyte cultures, resulting in depigmentation. Overexpressed microphthalmia-associated transcription factor was capable of rescuing the repressive effects of these compounds on the cotransfected tyrosinase promoter. Dark-skinned Yucatan swine treated with these agents showed visible skin lightening, which was confirmed histologically, whereas ultraviolet B-induced tanning of light-skinned swine was inhibited using these agents. Our findings suggest that modulation of microphthalmia-associated transcription factor expression can alter skin pigmentation and further confirm the central role of microphthalmia-associated transcription factor in melanogenesis.

Effect of a large deletion of the basic domain of mi transcription factor on differentiation of mast cells.

The mi transcription factor (MITF) is a basic-helix-loop-helix-leucine zipper transcription factor that is important for the development of mast cells. Cultured mast cells (CMCs) of mi/mi genotype express abnormal MITF (mi-MITF), but CMCs of tg/tg genotype do not express any MITFs. It was previously reported that mi/mi CMCs showed more severe abnormalities than tg/tg CMCs, indicating that mi-MITF had inhibitory function. Whereas mi-MITF contains a single amino acid deletion in the basic domain, MITF encoded by mi(ew) allele (ew-MITF) deletes 16 of 21 amino acids of the basic domain. Here the effect of a large deletion of the basic domain was examined. In mi(ew)/mi(ew) CMCs, the expression pattern of genes whose transcription was affected by MITF was comparable to that of tg/tg CMCs rather than to that of mi/mi CMCs. This suggested that ew-MITF lacked any functions. The part of the basic domain deleted in ew-MITF appeared necessary for either transactivation or inhibition of transactivation.

Linkage of M-CSF signaling to Mitf, TFE3, and the osteoclast defect in Mitf(mi/mi) mice.

Osteoclasts are multinucleated hematopoietic cells essential for bone resorption. Macrophage colony-stimulating factor (M-CSF) is critical for osteoclast development and function, although its nuclear targets in osteoclasts are largely unknown. Mitf and TFE3 are two closely related helix-loop-helix (HLH) transcription factors previously implicated in osteoclast development and function. We demonstrate that cultured Mitf(mi/mi) osteoclasts are immature, mononuclear, express low levels of TRAP, and fail to mature upon M-CSF stimulation. In addition, M-CSF induces phosphorylation of Mitf and TFE3 via a conserved MAPK consensus site, thereby triggering their recruitment of the coactivator p300. Furthermore, an unphosphorylatable mutant at the MAPK consensus serine is specifically deficient in formation of multinucleated osteoclasts, mimicking the defect in Mitf(mi/mi) mice. These results identify a signaling pathway that appears to coordinate cytokine signaling with the expression of genes vital to osteoclast development.

Microphthalmia transcription factor: a sensitive and specific marker for malignant melanoma in cytologic specimens.

BACKGROUND: The diagnosis of melanoma can be difficult because of shared cytomorphology with other malignant neoplasms. The most commonly used melanocytic markers, anti-S-100 protein and HMB-45 antigen, have limited specificity and sensitivity, respectively. Microphthalmia transcription factor (Mitf) is a nuclear transcription factor critical for the development and survival of melanocytes and has been shown as a sensitive and specific marker for melanoma in histologic specimens. METHODS: To evaluate the efficacy of Mitf as a marker for melanoma in cytologic preparations, 81 cell blocks from 44 patients with melanoma and 37 patients with nonmelanoma malignancies (29 patients with carcinoma, 4 patients with mesotheliomas, 2 patients with lymphoma, and 2 patients with islet cell tumors) were stained with monoclonal antibodies against Mitf (clone D5), S-100 protein, and HMB-45 antigen. The staining was evaluated blindly by three independent observers. The presence of nuclear staining for Mitf and cytoplasmic staining for S-100 protein or HMB-45 antigen in > 10% of tumor cells was considered positive staining for each antigen. RESULTS: Forty-four melanomas (100%), including all 3 spindle-cell melanomas, were positive for Mitf. All nonmelanoma neoplasms were negative with only one exception: One mammary carcinoma showed rare (< 10%), weak nuclear staining with Mitf. The sensitivity and specificity of Mitf as a marker for melanoma were both 100%, whereas the sensitivity of HMB-45 antigen was 90.4%, and the specificity of S-100 protein was 70.3%. CONCLUSIONS: Mitf is a sensitive and specific marker for malignant melanoma, including the spindle-cell variant, in cytologic specimens and may be superior to the current standard melanocytic markers, S-100 protein and HMB-45 antigen.

The generation and characterization of a cell line derived from a sporadic renal angiomyolipoma: use of telomerase to obtain stable populations of cells from benign neoplasms.

Angiomyolipomas are benign tumors of the kidney derived from putative perivascular epithelioid cells, that may undergo differentiation into cells with features of melanocytes, smooth muscle, and fat. To gain further insight into angiomyolipomas, we have generated the first human angiomyolipoma cell line by sequential introduction of SV40 large T antigen and human telomerase into human angiomyolipoma cells. These cells show phenotypic characteristics of angiomyolipomas, namely differentiation markers of smooth muscle (smooth muscle actin), adipose tissue (peroxisome proliferator-activator receptor gamma, PPARgamma), and melanocytes (microophthalmia, MITF), thus demonstrating that a single cell type can exhibit all of these phenotypes. These cells should serve as a valuable tool to elucidate signal transduction pathways underlying renal angiomyolipomas.

Microphthalmia transcription factor: not a sensitive or specific marker for the diagnosis of desmoplastic melanoma and spindle cell (non-desmoplastic) melanoma.

Microphthalmia transcription factor (Mitf), a melanocytic nuclear protein critical for the embryonic development and postnatal viability of melanocytes, is a master lineage regulator and modulates extracellular signals. Recently, Mitf expression was shown to be both a sensitive and specific marker of epithelioid melanoma. Because loss of specific melanocytic markers in melanomas with spindle cell morphology is more common compared with those tumors with epithelioid morphology, we investigated the sensitivity of D5, an anti-Mitf antibody, for diagnosis in this diagnostically problematic subset of melanomas. Twenty of 21 (95%) spindle cell and desmoplastic melanomas examined were reactive for S-100 protein. Only 4 of 21 (19%) spindle cell and desmoplastic melanomas were reactive for HMB-45. Six of 21 tumors (29%) were reactive for D5, including one case that was non-reactive for S-100 and HMB-45. Melan-A reactivity was seen in 2 of 13 cases (15%) studied. Eight of 24 (33%) non-melanocytic spindle cell tumors were reactive for D5, including 4 of 6 dermatofibromas, 1 of 6 schwannomas, 1 of 2 leiomyomas, and 2 of 6 leiomyosarcomas. Although D5 was shown in a previous study to be a highly sensitive and specific marker for epithelioid melanomas, the results of this study show it is not a sensitive or specific marker of spindle cell and desmoplastic melanomas. Nevertheless, we believe that diffuse positive staining for D5 when taken in clinical, histologic and immunohistochemical context may be diagnostically useful in selected cases of melanoma.

A CD44 survival pathway triggers chemoresistance via lyn kinase and phosphoinositide 3-kinase/Akt in colon carcinoma cells.

A major obstacle to successful treatment of colorectal cancer is chemotherapy resistance. Enhanced expression of variant CD44 isoforms has been associated with aggressive tumor behavior, prompting the question of whether signaling from this receptor might modulate drug sensitivity. Activation of variant CD44 in colon carcinoma cell lines triggered resistance to the drug 1,3-bis(2-chloroethyl)-1-nitrosurea. Resistance was induced by monoclonal antibodies directed against epitopes independent of the hyaluronate-binding region but was not triggered by identical treatment of a carcinoma line expressing the standard CD44 isoform. We observed that variant CD44 produced activation of the src-family tyrosine kinase lyn. Moreover, overexpression of dominant-active lyn recapitulated chemoresistance via a pathway shown to involve activation of phosphoinositide 3-kinase and Akt. These results establish a novel role for CD44 in determining survival of colon carcinoma cells through lyn kinase and Akt. The ability to suppress apoptosis might play a critical role in the onset and development of colorectal malignancies.

Linking osteopetrosis and pycnodysostosis: regulation of cathepsin K expression by the microphthalmia transcription factor family.

Various genetic conditions produce dysfunctional osteoclasts resulting in osteopetrosis or osteosclerosis. These include human pycnodysostosis, an autosomal recessive syndrome caused by cathepsin K mutation, cathepsin K-deficient mice, and mitf mutant rodent strains. Cathepsin K is a highly expressed cysteine protease in osteoclasts that plays an essential role in the degradation of protein components of bone matrix. Cathepsin K also is expressed in a significant fraction of human breast cancers where it could contribute to tumor invasiveness. Mitf is a member of a helix-loop-helix transcription factor subfamily, which contains the potential dimerization partners TFE3, TFEB, and TFEC. In mice, dominant negative, but not recessive, mutations of mitf, produce osteopetrosis, suggesting a functional requirement for other family members. Mitf also has been found-and TFE3 has been suggested-to modulate age-dependent changes in osteoclast function. This study identifies cathepsin K as a transcriptional target of Mitf and TFE3 via three consensus elements in the cathepsin K promoter. Additionally, cathepsin K mRNA and protein were found to be deficient in mitf mutant osteoclasts, and overexpression of wild-type Mitf dramatically up-regulated expression of endogenous cathepsin K in cultured human osteoclasts. Cathepsin K promoter activity was disrupted by dominant negative, but not recessive, mouse alleles of mitf in a pattern that closely matches their osteopetrotic phenotypes. This relationship between cathepsin K and the Mitf family helps explain the phenotypic overlap of their corresponding deficiencies in pycnodysostosis and osteopetrosis and identifies likely regulators of cathepsin K expression in bone homeostasis and human malignancy.

The p53 tumor suppressor: critical regulator of life & death in cancer.

p53 is the most commonly mutated or deleted known gene in human cancer. The consequences of its disruption are profound, either in the germlines of patients with Li-Fraumeni Syndrome, or in mice with targeted gene knockouts. Abundant evidence suggests that p53 exerts regulation of cell cycle progression as well as apoptotic cell death, both in response to identical environmental or metabolic stressors. The specific decision of cell cycle arrest vs. death may underlie p53's differential ability to trigger death in cancer cells and arrest with repair in non-cancer cells, thus producing a therapeutic index pertinent to cancer therapy. Indeed, p53 status is likely to correlate with prognosis in many human cancers and in multiple animal tumor models. The mechanistic basis for p53's functions are still emerging, and will hopefully yield new therapeutic strategies applicable to treatment of the many poor-prognosis, p53-deficient human malignancies.

Immunohistochemical and reverse transcription-polymerase chain reaction expression analysis of tyrosinase and microphthalmia-associated transcription factor in angiomyolipomas.

Angiomyolipomas (AMLs) show a characteristic immunoreactivity with melanocyte differentiation markers such as monoclonal antibody (mAb) HMB45, which detects melanocyte differentiation antigen gp100 and mAb A103 reacting with Melan-A/MART-1. Monoclonal antibody T311 to tyrosinase (a key enzyme of melanogenesis) and mAb D5 to the microphthalmia (Mitf) antigen are two newly available markers of melanocytic differentiation. The authors tested 15 AMLs with T311 and D5 by immunohistochemistry and a subset of 3 cases by reverse transcription-polymerase chain reaction for their expression of tyrosinase and Mitf mRNA. T311 showed poor sensitivity in AMLs because only focal staining was seen in 1 out of 15 cases, although tyrosinase mRNA was found in all tested cases. Mitf mRNA was present in 3 of 3 tested cases, and D5 was positive in 15 of 15 AMLs. However, D5 immunostaining often was focal and not as homogeneous as A103, which was analyzed in a previous study. D5 staining also could be seen in other cell types such as normal renal tubular cells, macrophages, and renal cell carcinoma. The current results show that in contrast with HMB45 and A103, T311 has little or no value in the diagnosis of AMLs. D5 may be useful in a panel of antibodies in the diagnosis of AMLs.

Clear cell sarcoma shows immunoreactivity for microphthalmia transcription factor: further evidence for melanocytic differentiation.

Microphthalmia transcription factor, a melanocytic nuclear protein critical for the embryonic development and postnatal viability of melanocytes, is a master regulator in modulating extracellular signals. Recently, microphthalmia transcription factor expression was shown to be both a sensitive and specific marker of epithelioid melanoma. We investigated the sensitivity of D5, an anti-microphthalmia transcription factor antibody, for diagnosis of clear cell sarcoma (also known as malignant melanoma of soft parts). Immunoreactivity in a nuclear pattern for D5 was present in 8 of 12 (75%) tumors. D5 staining was strong in three tumors, moderate in two, and weak in three. S-100 protein expression was seen in all 12 cases that had clear cell sarcoma examined. HMB-45 staining was seen in 11 of 12 (92%) tumors. Focal Melan-A positivity was seen in 3 of 7 (43%) tumors. Although D5 was shown in a previous study to be a highly sensitive and specific marker for epithelioid melanomas, the results of this study expand the spectrum of tumors showing immunoreactivity for D5. D5 immunoreactivity in clear cell sarcoma provides further evidence for melanocytic differentiation in this unusual tumor.

Key targets for the execution of radiation-induced tumor cell apoptosis: the role of p53 and caspases.

In many human hematologic and solid malignancies, intrinsic or acquired treatment resistance remains a major obstacle for successful cancer therapy. The molecular understanding of how tumor cells respond to chemotherapy and ionizing radiation is rapidly evolving. Induction of programmed cell death, apoptosis, is one important strategy for successful cancer therapy. This has been shown convincingly for oncogene-transformed normal cells as well as tumor cells of lymphoid origin. However, the relevance of apoptosis in solid human malignancies is less clear. Loss of apoptosis might be linked to specific mutations in the often tissue-specific apoptotic pathways due to aberrations in the stress-related signal transduction cascades. Restoration of a dysfunctional apoptotic program in cancer tissue where apoptosis has been identified as an important mechanism for tissue homeostasis is one rational approach for innovative cancer therapy. In this review, we focus on the relevance of the tumor suppressor p53 for apoptosis-induction and successful cancer therapy outlining the importance of an intact caspase machinery for apoptosis execution. Strategies are discussed to overcome treatment resistance and a high apoptotic threshold in human malignancies where apoptosis is the dominant mode of cell death and the status of p53 is an important determinant for apoptosis induction.

Microphthalmia transcription factor expression in cutaneous benign, malignant melanocytic, and nonmelanocytic tumors.

The protein encoded by the microphthalmia (mi) gene is a transcription factor essential for the development and survival of melanocytes. Using a monoclonal antibody generated against human Mi transcription factor protein (Mitf) the authors previously demonstrated that Mitf expression is conserved in primary and metastatic malignant melanomas, and appears to be a highly sensitive and specific melanocytic marker. Mitf expression in various cutaneous nevi and cutaneous nonmelanocytic tumors has not been documented systematically. The authors evaluated Mitf immunostaining in 62 benign nevi, 58 primary cutaneous melanomas, and 53 nonmelanocytic tumors. Mitf immunostaining was conserved in all benign nevi, with Spitz nevi and neurotized nevi demonstrating decreased staining intensity. With the exception of desmoplastic melanomas, all primary cutaneous melanomas were immunopositive regardless of the cell type. Only one of 14 desmoplastic melanomas was Mitf positive. None of the nonmelanocytic tumors was immunopositive, including those lesions that may resemble melanoma histologically (spindle cell carcinomas, atypical fibroxanthomas, and leiomyosarcomas). The results demonstrate that Mitf antibody expression is conserved in the majority of benign and malignant melanocytic lesions, and that it may be helpful in the diagnosis of primary melanocytic skin lesions. Its use in desmoplastic melanomas is limited and is reflective of other melanocyte-associated antigens. Mitf discriminates between spindle cell nonmelanocytic tumors and melanomas with a spindle cell morphology, and is useful in a panel with other appropriate antibodies.