Genetic alterations that activate the mitogen-activated protein kinase (MAP kinase) pathway occur commonly in cancer. For example, the majority of melanomas harbor mutations in the BRAF oncogene, which are predicted to confer enhanced sensitivity to pharmacologic MAP kinase inhibition (e.g., RAF or MEK inhibitors). We investigated the clinical relevance of MEK dependency in melanoma by massively parallel sequencing of resistant clones generated from a MEK1 random mutagenesis screen in vitro, as well as tumors obtained from relapsed patients following treatment with AZD6244, an allosteric MEK inhibitor. Most mutations conferring resistance to MEK inhibition in vitro populated the allosteric drug binding pocket or ␣-helix C and showed robust (Ϸ100-fold) resistance to allosteric MEK inhibition. Other mutations affected MEK1 codons located within or abutting the Nterminal negative regulatory helix (helix A), which also undergo gain-of-function germline mutations in cardio-facio-cutaneous (CFC) syndrome. One such mutation, MEK1(P124L), was identified in a resistant metastatic focus that emerged in a melanoma patient treated with AZD6244. Both MEK1(P124L) and MEK1(Q56P), which disrupts helix A, conferred cross-resistance to PLX4720, a selective B-RAF inhibitor. However, exposing BRAF-mutant melanoma cells to AZD6244 and PLX4720 in combination prevented emergence of resistant clones. These results affirm the importance of MEK dependency in BRAF-mutant melanoma and suggest novel mechanisms of resistance to MEK and B-RAF inhibitors that may have important clinical implications.BRAF ͉ drug resistance ͉ MAP kinase ͉ melanoma A pproximately one-third of all cancers harbor genetic alterations that aberrantly upregulate mitogen-activated protein kinase (MAPK)-dependent signal transduction (1). In the MAPK pathway, RAS oncoproteins activate RAF, MEK, and ERK kinases to direct key cell proliferative and survival signals. When rendered constitutively active by genetic mutation, the MAP kinase pathway is believed to confer ''oncogene dependency'' (2), an excessive reliance on its dysregulated activity for tumor viability. Therefore, protein kinases within this signaling cascade offer promising targets for novel anticancer therapeutics.In melanoma, uncontrolled MAP kinase pathway activity is nearly ubiquitous and occurs most commonly through gain-offunction mutations involving codon 600 of the B-RAF kinase (3) (BRAF V600E ; 50-70% of cases). Considerable preclinical evidence has associated the BRAF V600E mutation with heightened sensitivity to pharmacologic inhibition of RAF or MEK kinases (4, 5). Although early clinical trials of RAF and MEK inhibitors failed to show a substantial benefit (6, 7), recent phase I studies of selective RAF inhibitors have shown promising results in patients with BRAF-mutant tumors (8, 9). Thus, optimizing therapeutic efficacy while avoiding or bypassing the emergence of resistance to MAP kinase pathway inhibition will likely gain increasing importance in melanoma and other MAP kinasedriven cancers.He...

A B S T R A C TPurpose RAF inhibitors are effective against melanomas with BRAF V600E mutations but may induce keratoacanthomas (KAs) and cutaneous squamous cell carcinomas (cSCCs). The potential of these agents to promote secondary malignancies is concerning. We analyzed cSCC and KA lesions for genetic mutations in an attempt to identify an underlying mechanism for their formation. MethodsFour international centers contributed 237 KA or cSCC tumor samples from patients receiving an RAF inhibitor (either vemurafenib or sorafenib; n ϭ 19) or immunosuppression therapy (n ϭ 53) or tumors that developed spontaneously (n ϭ 165). Each sample was profiled for 396 known somatic mutations across 33 cancer-related genes by using a mass spectrometric-based genotyping platform. ResultsMutations were detected in 16% of tumors (38 of 237), with five tumors harboring two mutations. Mutations in TP53, CDKN2A, HRAS, KRAS, and PIK3CA were previously described in squamous cell tumors. Mutations in MYC, FGFR3, and VHL were identified for the first time. A higher frequency of activating RAS mutations was found in tumors from patients treated with an RAF inhibitor versus populations treated with a non-RAF inhibitor (21.1% v 3.2%; P Ͻ .01), although overall mutation rates between treatment groups were similar (RAF inhibitor, 21.1%; immunosuppression, 18.9%; and spontaneous, 17.6%; P ϭ not significant). Tumor histology (KA v cSCC), tumor site (head and neck v other), patient age (Յ 70 v Ͼ 70 years), and sex had no significant impact on mutation rate or type. ConclusionSquamous cell tumors from patients treated with an RAF inhibitor have a distinct mutational profile that supports a mechanism of therapy-induced tumorigenesis in RAS-primed cells. Conceivably, cotargeting of MEK together with RAF may reduce or prevent formation of these tumors.

Interferons (IFNs) are cytokines that possess potent anti-viral and immunoregulatory activities. In contrast, their potential role(s) in anti-bacterial defense and neutrophil activation mechanisms is less well explored. By comparing gene expression patterns between immature and mature human neutrophils, we obtained evidence that intracellular proteases and other anti-bacterial proteins are produced at earlier stages of maturation, whereas the genes for receptors and signaling molecules required for the release of these effector molecules are preferentially induced during terminal differentiation. For instance, mature neutrophils strongly expressed genes that increase their responses to type I and type II IFNs. Interestingly, granulocyte/macrophage colonystimulating factor was identified as a repressor of IFN signaling components and consequently of IFN-responsive genes. Both IFN-␣ and IFN-␥ induced strong tyrosine phosphorylation of STAT1 in mature but not in immature neutrophils. Functional in vitro studies suggested that IFNs act as priming factors on mature neutrophils, allowing the formation of extracellular traps upon subsequent stimulation with complement factor 5a (C5a). In contrast, both IFN-␣ and IFN-␥ had only little capacity to prime immature cells in this system. Moreover, both IFNs did not have significant anti-proliferative effects on immature neutrophils. These data contribute to our understanding regarding changes of gene expression during neutrophil differentiation and IFNmediated anti-bacterial defense mechanisms.

Therapeutic SNRB is effective in sciatica, but early response does not predict the effect after 2 weeks. Type 1 injections are more painful than type 2 injections.

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Plasmid DNA encoding human interleukin 12 (IL-12) was produced under GMP conditions and injected into lesions of nine patients with malignant melanoma (stage IV) previously treated with both standard and nonstandard therapies. The treatment was based on efficacy in preclinical studies with melanoma in mice and gray horses. The DNA was applied in cycles, three injections per cycle, for up to seven cycles. Three therapy arms comprised low (2 mg), medium (4 mg), and high (10 to 20 mg) amounts of total DNA. The therapy was well tolerated. Three of nine patients experienced a clinical response: two stable disease and one complete remission. One patient receiving a low dose of DNA experienced a long-lasting stabilization of the disease for more than 3 years, whereas the other two responders received high doses of DNA. All patients but one (patient 9) experienced a transient response at the intratumoral injection site. Immunohistochemical staining of responder sections showed local reduction of angiogenesis and lymphocyte infiltrations. All patients, in particular the clinical and local responders (patients 3, 7, and 8), exhibited an antigen-specific immune response against MAGE-1 and MART-1, which in some cases preexisted. Biopsies of responders showed some increase in IL-12, IP-10, and IFN-(). Serum levels revealed fluctuations. The results show that intratumoral injection of DNA produced some beneficial clinical effect. DNA encoding a cytokine may be useful as a therapeutic or adjuvant against various human cancers.

Cutaneous T-cell lymphomas (CTCLs) are malignancies of skin-homing lymphoid cells, which have so far not been investigated thoroughly for common oncogenic mutations. We screened 90 biopsy specimens from CTCL patients (41 mycosis fungoides, 36 Sé zary syndrome, and 13 non-mycosis fungoides/Sé zary syndrome CTCL) for somatic mutations using OncoMap technology. We detected oncogenic mutations for the RAS pathway in 4 of 90 samples. One mycosis fungoides and one pleomorphic CTCL harbored a KRAS G13D mutation; one Séz-ary syndrome and one CD30 ؉ CTCL harbored a NRAS Q61K amino acid change. All mutations were found in stage IV patients (4 of 42) who showed significantly decreased overall survival compared with stage IV patients without mutations (P ‫؍‬ .04). In addition, we detected a NRAS Q61K IntroductionCutaneous T-cell lymphomas (CTCLs) are rare malignancies of skin-homing T lymphocytes. Curative modalities have thus far proven elusive. CTCL microarray studies have revealed natural clusters in association with prognosis. 1 Array-based comparative genomic hybridization (CGH) combined with gene expression profiling identified highly recurrent chromosomal alterations both in mycosis fungoides (MF) and Sézary syndrome (SS) patient specimens. 2,3 For example, FASTK and SKAP1 gene loci showed recurrent gains, and these genes also exhibited increased expression, whereas RB1 and DLEU tumor suppressor genes displayed diminished expression associated with loss. In another study, recurrent deletion of tumor suppressor genes BCL7A, SMAC/ DIABLO, and RHOF in MF was observed. 4 Genomic patterns characteristic of MF differ markedly from SS. 5 This might implicate discriminative molecular pathogenesis and different therapeutic requirements.The RAS-RAF-MEK-ERK signaling pathway regulates cell responses to environmental stimuli and plays a crucial role in many cancers. 6 Thus, RAF and MEK are attractive therapeutic targets. 7,8 RAS is a small guanine-nucleotide binding protein that is attached to the inner side of the plasma membrane. Activation of RAS causes RAF recruitment and activation by phosphorylation. Activated RAF kinase phosphorylates and activates MEK, which phosphorylates ERK. Three RAS (KRAS, NRAS, and HRAS), 3 RAF (ARAF, BRAF, and CRAF), 2 MEK (MEK1 and MEK2), and 2 ERK (ERK1 and ERK2) isoforms compose the "canonical" mitogen-activated protein kinase pathway. Somatic mutations that are found in many cancers, including colon carcinoma, melanoma, or pancreatic cancer, occur almost exclusively in BRAF, KRAS, or NRAS isoforms. 9-11 Typical mutations affect glycine 12 (G12), glycine 13 (G13), or glutamine 61 (Q61) and keep RAS in an activated form. The RAS pathway regulates survival, proliferation, senescence, and differentiation. However, in tumor cells, mutated (oncogenic) RAS preferentially promotes survival and proliferation. Thus, RAF and MEK kinases serve as suitable drug targets. RAF is targeted by inhibitors in preclinical or clinical development, including, for example, RAF265 and PLX4720. 12,13 However, target...

Lymph node (LN) status is an important prognostic factor in melanoma patients. p16 expression and proliferation rate (MIB-1) of primary melanomas have been suggested as a marker of metastatic potential. In this study, the correlation of p16 expression and the proliferation rate (MIB-1) with LN status and tumor-specific survival was investigated in primary melanomas. MIB-1 and p16 expression were analyzed by immunohistochemistry in 64 patients with primary cutaneous melanoma. Thirty four nevi were used as control. All patients underwent sentinel lymph node staging. Three different p16 staining patterns were observed: a combination of nuclear and cytoplasmic staining, only cytoplasmic staining and absence of p16 expression. All 34 nevi displayed a nuclear and cytoplasmic p16 staining, whereas p16 was negative in 14 of 64 (22%) melanomas. The level of p16 expression gradually decreased from benign nevi to melanoma without metastasis to melanoma with metastasis. There was a significant correlation between cytoplasmic p16 expression and absence of metastasis (p < 0.05). Death of disease correlated with absence of p16 immunostaining (p 5 0.01). MIB-1 expression was not associated with survival. These results confirm the relevance of p16 expression as a prognostic marker in melanoma patients. In addition, it was shown that cytoplasmic immunostaining for p16 in primary melanoma might serve as a predictor of the LN status. Therefore, immunohistochemical evaluation for p16 expression is of potential value for treatment planning in melanoma surgery. ' 2005 Wiley-Liss, Inc.Key words: melanoma; nevus; p16; sentinel lymph node; MIB-1 Cutaneous malignant melanoma remains the leading cause of skin cancer death in industrialized countries. Up to date, the only possible therapy to cure melanoma patients is surgical excision of localized, nonmetastatic primary cutaneous melanoma (stage I and stage II patients).1 Unfortunately, 20% of clinically stage I and II patients already have micrometastatic disease at diagnosis. Currently, there is no cure for patients who present with visceral melanoma metastases. Therefore, identifying patients at increased risk for metastases is one of the most critical issues in the management of melanoma. These patients have to be considered for adjuvant therapy.Several prognostic factors have been identified. These include age, gender, anatomical site, 2 Breslow tumor thickness, 3-5 ulceration, mitotic rate, 6 tumor infiltrating lymphocytes, 7 vascular invasion and metastasis. [8][9][10][11] Recent studies have identified varies molecular markers as additional alterations for the metastatic potential of malignant melanoma. 12The product of the p16/INK4a/CDKN2/MTS tumor suppressor gene acts as a negative cell cycle regulator by inhibiting G1 cyclin-dependent kinases, which are phosphorylating the retinoblastoma protein.13,14 p16 is inactivated by mutation, promotor methylation or loss of heterozygosity in a wide range of human malignancies, 15,16 including familial and sporadic melanoma. 17,18Furthermo...