Prognostic markers in lentigo maligna patients treated with imiquimod cream: A long-term follow-up study.

BACKGROUND: More data are needed to define factors that predict long-term success after imiquimod therapy for lentigo maligna (LM). OBJECTIVE: We sought to determine the demographic, clinical, and histologic prognostic markers of relapse-free survival in patients with LM who were treated with imiquimod. METHODS: This was a single-arm, open-label, nonrandomized, prospective study. RESULTS: Eighty-nine patients with histologically confirmed LM and a median follow-up time of 4.8 years after imiquimod treatment were included in our study. Sixteen patients (18%) relapsed. Statistically significant indicators of an increased risk of local recurrence included: the total number of melanocytes, the number of basal and suprabasal melanocytes and the number of pagetoid spreading melanocytes. LIMITATIONS: Our study was a single-center, nonrandomized study. CONCLUSION: An assessment of different melanocyte fractions in the diagnostic baseline biopsy specimen may help to predict the response of LM to imiquimod therapy.

Localized Epidermal Cysts as a Radiation Recall Phenomenon in a Melanoma Patient Treated with Radiotherapy and the BRAF Inhibitor Vemurafenib.

BRAF inhibitors are broadly used for metastatic melanoma with BRAF mutations. Their use results in various cutaneous side effects, such as the development of keratoacanthomas and squamous cell carcinomas. We report a patient with metastatic melanoma treated with vemurafenib who developed dozens of histologically confirmed epidermal cysts within 2 months after initiation of vemurafenib administration. The cystic lesions were observed only in the localized area where a large exophytic melanoma tumor mass had been previously irradiated. Localized epidermal cysts may constitute an unusual radiation recall reaction in patients treated with BRAF inhibitors.

Characterization of the DNA copy-number genome in the blood of cutaneous T-cell lymphoma patients.

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous non-Hodgkin's lymphoma that may variably involve the skin, lymph nodes, and peripheral blood. Malignant burden ranges from cutaneous patches and plaques with little evidence of blood involvement to erythroderma often in association with frank leukemia, as in Sézary syndrome. Toward a better understanding of the pathogenesis of this CD4+ T-cell malignancy, we conducted a high-resolution genomic analysis combining DNA (23 samples) and mRNA (12 samples) data of peripheral blood isolates from CTCL patients across a spectrum of stages. Strikingly, even patients with limited involvement, e.g., normal CD4 counts, contained significant copy-number alterations. Defining genomic characteristics of CTCL blood involvement included gains on 8q and 17q, and deletions on 17p and chromosome 10. A consensus analysis of 108 leukemic CTCL samples demonstrated global similarities among patients with varied blood involvement, narrowing 38 of 62 loci. Toward an annotated framework for in vitro testing, we also characterized genomic alterations in five CTCL cell lines (HH, HUT78, PNO, SeAx, and Sez4), revealing intact core features of leukemic CTCL. Together, these studies produce the most comprehensive view of the leukemic CTCL genome to date, with implications for pathogenesis, molecular classification, and potential future therapeutic developments.

RAS mutations are associated with the development of cutaneous squamous cell tumors in patients treated with RAF inhibitors.

PURPOSE: 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. METHODS: Four international centers contributed 237 KA or cSCC tumor samples from patients receiving an RAF inhibitor (either vemurafenib or sorafenib; n = 19) or immunosuppression therapy (n = 53) or tumors that developed spontaneously (n = 165). Each sample was profiled for 396 known somatic mutations across 33 cancer-related genes by using a mass spectrometric-based genotyping platform. RESULTS: Mutations 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 < .01), although overall mutation rates between treatment groups were similar (RAF inhibitor, 21.1%; immunosuppression, 18.9%; and spontaneous, 17.6%; P = not significant). Tumor histology (KA v cSCC), tumor site (head and neck v other), patient age (≤ 70 v > 70 years), and sex had no significant impact on mutation rate or type. CONCLUSION: Squamous 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.

High-throughput mutation profiling of CTCL samples reveals KRAS and NRAS mutations sensitizing tumors toward inhibition of the RAS/RAF/MEK signaling cascade.

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 Sézary syndrome, and 13 non-mycosis fungoides/Sézary 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 Sézary 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) mutation in the CTCL cell line Hut78. Knockdown of NRAS by siRNA induced apoptosis in mutant Hut78 cells but not in CTCL cell lines lacking RAS mutations. The NRAS(Q61K) mutation sensitized Hut78 cells toward growth inhibition by the MEK inhibitors U0126, AZD6244, and PD0325901. Furthermore, we found that MEK inhibitors exclusively induce apoptosis in Hut78 cells. Taken together, we conclude that RAS mutations are rare events at a late stage of CTCL, and our preclinical results suggest that such late-stage patients profit from MEK inhibitors.

Melanoma after laser therapy of pigmented lesions--circumstances and outcome.

The use of laser therapy in the treatment of pigmented lesions is a controversial issue as it can delay melanoma diagnosis and may negatively impact mortality. Few cases of melanoma after laser therapy have been reported. It is still unknown whether melanoma can be induced by lasers. We discuss the outcomes of twelve patients presenting with melanoma subsequent to previous treatment with laser. In four patients, a skin biopsy was performed before laser treatment. Histology was re-evaluated by a panel of experienced dermatopathologists and analyzed in the context of clinical and photo-optical data. There was evidence for pathological misdiagnosis in two cases. The other two cases initially presented with non-suspicious features before laser treatment and were clearly diagnosed as melanoma thereafter, opening the possibility of melanoma induction by laser treatment. Most patients were female and presented with facial lesions. Three patients have already died of melanoma and two are in stage IV, showing progressive disease with distant metastases. Laser therapy is a common treatment for pigmented lesions, increasing the risk of delayed melanoma diagnosis. This prevents appropriate and timely therapy, and may therefore lead to a fatal outcome. A careful examination of all pigmented lesions using surface microscopy and representative biopsies in combination with a close follow-up is recommended.

MEK1 mutations confer resistance to MEK and B-RAF inhibition.

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 alpha-helix C and showed robust ( approximately 100-fold) resistance to allosteric MEK inhibition. Other mutations affected MEK1 codons located within or abutting the N-terminal 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.

Granulomatous slack skin responds to UVA1 phototherapy.

Granulomatous slack skin (GSS) is an extremely rare disorder within the group of cutaneous T cell lymphomas (CTCL). Ultraviolet A1 (UVA1) phototherapy has previously been reported to be useful in the treatment of CTCL such as mycosis fungoides. We report a 35-year-old Caucasian male with GSS treated with UVA1 phototherapy starting at 20 J/cm(2) UVA1 3 times a week and subsequently increased in increments of 5 J/cm(2) to a medium-range dose of 50 J/cm(2) per session. The patient underwent a total of 45 sessions with a cumulative dose of 1,495 J/cm(2) UVA1 without any adverse events. At the conclusion of UVA1 phototherapy, a decrease in erythema and skin thickness was observed which was most prominent in the periphery of the lesion in the right groin area. A follow-up 12 months after phototherapy showed continued treatment benefit. To our knowledge, this is the first report describing the successful use of UVA1 (340-400 nm) phototherapy in a patient with GSS.

Baseline staging of melanoma with unknown primary site: the value of serum s100 protein and positron emission tomography.

BACKGROUND: Baseline staging is important in all melanoma types, including melanoma with unknown primary site (MUP). Staging includes different examination strategies, each with different accuracy. OBJECTIVE: To determine the value of serum S100 protein levels and positron emission tomography (PET) in the baseline staging of MUP. METHODS: Twenty patients with MUP were evaluable for the analysis between 1996 and 2007 with both S100 assessment and PET performed for baseline staging. RESULTS: Serum S100 was elevated in 7 patients (35%). The PET scan detected the metastases in 6 of 7 patients with elevated serum S100 protein showing a strong correlation (p = 0.005). Patients with metastases had significantly higher serum S100 levels (p = 0.01) than the ones without. Serum S100 protein was shown to be discriminative between patients with and without metastases (receiver-operating characteristic, p = 0.012) with 75% sensitivity and 92% specificity. CONCLUSION: Serum S100 protein appears to be a sensitive as well as specific marker to detect metastases. We therefore might recommend serum S100 assessment to be included in the baseline staging of MUP.

Skin problems associated with pegylated liposomal doxorubicin-more than palmoplantar erythrodysesthesia syndrome.

Liposomal pegylated doxorubicin is an encapsulation form of doxorubicin, with an improved pharmacokinetic profile and the ability to selectively accumulate into tumor tissue. As a result, the tolerated dose of the drug can be increased, followed by a reduced incidence of neutropenia and cardiotoxicity in comparison to doxorubucin treatment. However, a common adverse dose-schedule limiting effect of the treatment is palmoplantar erythrodysesthesia syndrome. In this retrospective study we included six patients hospitalised in the University Hospital of Zurich during the last 2 years, in connection with side effects caused by pegylated liposomal doxorubicin. These patients received this chemotherapeutic agent for treatment of various malignancies such as breast cancer, ovarian cancer, mycosis fungoides and cutaneous B-cell lymphoma. Three of six patients in this study developed classical palmoplantar erythrodysesthesia, one developed palmoplantar erythrodysesthesia associated with extensive bullous disease, one developed eruption of lymphocyte recovery syndrome and one developed intertrigo like dermatitis with stomatitis. Pegylated liposomal doxorubicin induces various skin reactions including palmoplantar erythrodysesthesia syndrome. However, the exact clinical presentation might depend on pre-existing skin diseases.

Reduced pSmad2 immunodetection correlates with increased primary melanoma thickness.

Cutaneous melanoma is the most aggressive of cutaneous neoplasms. Identifying patients with an increased risk for the development of metastases is critical. This study investigates phospho-Smad2, a central factor of the transforming growth factor beta pathway, on formalin-fixed, paraffin-embedded tissues from 60 primary cutaneous melanomas (Breslow >1 mm), for its candidacy for being a prognostic marker in primary cutaneous melanoma. Phospho-Smad2 positivity was assessed for correlation with clinical parameters including Breslow index, melanoma type, survival, development of metastases, sentinel lymph node status and age. Phospho-Smad2 positivity was not associated with survival or development of metastases, suggesting that it would not be a useful prognostic marker. Despite this, we found phospho-Smad2 positivity to be correlated with low tumour thickness, indicating that as the primary tumour grows there is an increased inhibition of transforming growth factor beta signalling resulting in suppressed Smad2 phosphorylation. Additionally, phosphorylation of Smad2 in neighbouring melanoma cells and keratinocytes was interrelated, which is a further indication that Smad2 phosphorylation in primary melanoma is affected by local area microenvironmental factors. We hypothesize that the observed decrease in transforming growth factor beta signalling in thicker primary melanomas is due to the increased production of signalling inhibitors.

Recombinant measles virus induces cytolysis of cutaneous T-cell lymphoma in vitro and in vivo.

Measles virus (MV) has shown promise as an oncolytic virus in the treatment of different tumor models for human B-cell lymphoma, multiple myeloma, ovarian cancer, and glioma. We have shown that, in a phase I clinical trial, MV vaccine induces tumor regression in cutaneous T-cell lymphoma (CTCL) patients. Here, we investigated in detail, the effect of recombinant MV (rMV) vaccine strain in CTCL cell cultures, and in vivo in established CTCL xenografts in nude mice. The susceptibility of three CTCL cell lines, originating from patients, to rMV was tested by determination of cell surface expression of MV receptors. All cell lines expressed the receptors CD150 and CD46 and were easily infected by rMV and induced complete cell lysis. The cytoreductive activity was apparent in cells forming aggregates, indicating a cell-to-cell spread of MV and cytolysis owing to virus infection. Intratumoral (i.t.) injection of rMV, expressing enhanced green fluorescent protein induced complete regression of large established human CTCL tumors in nude mice, whereas tumors with control treatment progressed exponentially. Immunohistochemical analysis of tumor biopsies, after i.t. treatment, for MV-NP protein complex demonstrated replication of MV within the tumors. The data demonstrate the potential of MV as a therapeutic agent against CTCL.

p16 expression in primary malignant melanoma is associated with prognosis and lymph node status.

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 = 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.

Oncolytic measles virus in cutaneous T-cell lymphomas mounts antitumor immune responses in vivo and targets interferon-resistant tumor cells.

Some cutaneous T-cell lymphomas, (CTCLs) clonal T cells are deficient in interferon signaling, making them promising targets for viral oncolysis. We evaluated cytopathic effects of measles virus (MV) in CTCL. CTCL cell lines and infiltrating lymphocytes in CTCL expressed MV receptors CD150 and CD46. In a phase 1 dose escalation trial a total of 16 injections of live MV, Edmonston-Zagreb vaccine strain, were given intratumorally to 5 patients with CTCL. Patients had antimeasles-serum antibodies and were pretreated with interferon-alpha to prevent uncontrolled virus spread. The well-tolerated treatment with MV resulted in clinical responses. Evaluation of biopsies, before and at 11 days after injection, by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated local viral activity with positive staining for MV nucleoprotein (NP), an increase of the interferon gamma (IFN-gamma)/CD4 and IFN-gamma/CD8 mRNA ratios and a reduced CD4/CD8 ratio. All patients demonstrated an increased antimeasles antibody titer after therapy. The data demonstrate that CTCLs are promising targets for an MV-based oncolytic therapy.

Intratumoral injection of DNA encoding human interleukin 12 into patients with metastatic melanoma: clinical efficacy.

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.

Imiquimod treatment induces expression of opioid growth factor receptor: a novel tumor antigen induced by interferon-alpha?

PURPOSE: Imiquimod represents a synthetic local immune response modifier that has demonstrated efficacy in clearing basal cell carcinoma. Via interaction with Toll-like receptor 7 on immune cells, imiquimod induces local production of cytokines, such as interferon (IFN)-alpha. EXPERIMENTAL DESIGN: To more closely define and elucidate mechanisms leading to basal cell carcinoma clearance in vivo, we examined gene expression profiles of skin basal cell carcinoma before and after treatment with 5% imiquimod cream (Aldara) by using high-density oligonucleotide arrays. RESULTS: We show that imiquimod predominantly induces genes involved in different aspects of immune response. In addition to effects on immunity, imiquimod treatment modulates the expression of genes involved in the control of apoptosis and oncogenesis. Array data indicated that imiquimod treatment induces expression of opioid growth factor receptor, a molecule recently reported to be a target for antitumor antibody responses. Immunohistochemistry revealed in vivo up-regulation of opioid growth factor receptor protein on tumor and on infiltrating cells after treatment. By using basal cell carcinoma cell lines treated with IFN-alpha or imiquimod, we show that opioid growth factor receptor up-regulation is IFN-alpha-mediated, rather then directly imiquimod-mediated. By using tissue microarray containing 52 basal cell carcinomas, we demonstrate opioid growth factor receptor expression in almost half of the cases. Expression of opioid growth factor receptor correlated with a longer recurrence-free period in basal cell carcinoma that recurred after radiotherapy (Kaplan-Meier analysis, P = 0.041). CONCLUSIONS: In addition to its immunomodulatory and antiproliferative activity, opioid growth factor receptor seems to have a prognostic significance in basal cell carcinoma patients. Our data add to the growing list of basal cell carcinoma-associated tumor antigens.

Establishment of a mouse xenograft model for mycosis fungoides.

Mycosis fungoides (MF) is the most frequent variant of cutaneous T-cell lymphomas (CTCLs). MF primarily involves the skin initially with patches and plaques. In later stages, cutaneous tumors develop and tumor cells may spread to lymph nodes and finally to visceral sites. Here, we describe an animal model for MF in immune-deficient nude mice, using the CTCL cell line MyLa. Subcutaneous transplantation of MyLa cells leads to the formation of cutaneous tumors in 80% of the mice (50/60 total). Spread of tumor cells to visceral sites was detected by immunohistochemistry and polymerase chain reaction (PCR)-based detection of specific T-cell receptor-gamma rearrangement. MyLa cells were found circulating in the blood, lymph nodes, and in blood vessels of heart, kidney, lung, and liver. In lung and liver tissue, tumor cells presented perivascular invasion, but no large secondary tumors developed. The nude mouse model described here will be a valuable test system for new therapeutic approaches for the treatment of MF and opens the unique opportunity to study the disease in vivo.