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 α-helix C and showed robust (almost equal to100-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.
Summary
There is growing evidence that the metastatic spread of melanoma is driven not by a linear increase in tumorigenic aggressiveness, but rather by switching back and forth between two different ...phenotypes of metastatic potential. In vitro these phenotypes are respectively defined by the characteristics of strong proliferation/weak invasiveness and weak proliferation/strong invasiveness. Melanoma cell phenotype is tightly linked to gene expression. Taking advantage of this, we have developed a gene expression–based tool for predicting phenotype called Heuristic Online Phenotype Prediction. We demonstrate the predictive utility of this tool by comparing phenotype‐specific signatures with measurements of characteristics of melanoma phenotype‐specific biology in different melanoma cell lines and short‐term cultures. We further show that 86% of 536 tested melanoma lines and short‐term cultures are significantly associated with the phenotypes we describe. These findings reinforce the concept that a two‐state system, as described by the phenotype switching model, underlies melanoma progression.
Purpose: Raf/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signaling pathway is constitutively
activated in melanoma. AZD6244 blocks MEK1/2, inhibiting ERK ...phosphorylation. We focus on associated cutaneous toxicity and
we attempt to understand the underlying pathophysiology and design treatment strategies.
Experimental Design: Dermatologic conditions of 22 patients with unresectable melanoma stage III/IV in a phase II trial were evaluated. Thirteen
patients received AZD6244 initially, and nine patients were treated with AZD6244 following tumor progression with temozolomide.
Biopsies were compared with matched controls in normal skin. Immunohistochemistry was performed. Half-side treatment of acute
skin toxicity compared therapeutic options.
Results: Nineteen of 22 (86%) AZD6244-treated patients presented with cutaneous eruptions. Seventeen patients (77%) developed acute
papulopustular rash. Chronic skin changes included xerosis, paronychia, and fissured fingertips, resembling cutaneous toxicity
of epidermal growth factor receptor inhibition. In addition, we observed reduced pigmentation of hair and skin. Histology
of acute skin lesions revealed a significant increase of apoptotic keratinocytes ( P = 0.0008), focal neutrophilic infiltrates, destruction of the adnexal structures by neutrophils, and reduced cytokeratins.
A significant proliferation shift from basal to suprabasal keratinocytes was shown in acute and chronic lesions. The number
and viability of melanocytes was not affected. Corticosteroids plus antibacterial topical therapy ameliorate acute skin toxicity.
Conclusions: AZD6244-associated skin reactions partly overlap with those observed upon epidermal growth factor receptor inhibition. Additionally,
pigmentation of skin and hair is affected. The interruption of the MEK signaling pathway results in an acute keratinocyte
stress response with disturbed epidermal homeostasis, inflammation, and tissue damage. Chronic adaptation controls inflammatory
tissue damage but leads to cutaneous malfunctions that explain chronic skin toxicity. Clin Cancer Res; 16(3); 1058–64
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 KRASG13D mutation; one Sézary syndrome and one CD30+ CTCL harbored a NRASQ61K 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 NRASQ61K 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 NRASQ61K 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.
Summary
Recent observations suggest that melanoma cells drive disease progression by switching back and forth between phenotypic states of proliferation and invasion. Phenotype switching has been ...linked to changes in Wnt signalling, and we therefore looked for cell phenotype‐specific differences in the levels and activity of β‐catenin and its LEF/TCF co‐factors. We found that while cytosolic β‐catenin distribution is phenotype‐specific (membrane‐associated in proliferative cells and cytosolic in invasive cells), its nuclear distribution and activity is not. Instead, the expression patterns of two β‐catenin co‐factors, LEF1 and TCF4, are both phenotype‐specific and inversely correlated. LEF1 is preferentially expressed by differentiated/proliferative phenotype cells and TCF4 by dedifferentiated/invasive phenotype cells. Knock‐down experiments confirmed that these co‐factors are important for the phenotype‐specific expression of M‐MITF, WNT5A and other genes and that LEF1 suppresses TCF4 expression independently of β‐catenin. Our data show that melanoma cell phenotype switching behaviour is regulated by differential LEF1/TCF4 activity.
Summary
Oncogenic mutations within the MAPK pathway are frequent in melanoma, and targeting of MAPK signaling has yielded spectacular responses in a significant number of patients that last for ...several months before relapsing. We investigated the effects of two different inhibitors of MAPK signaling in proliferative and invasive melanoma cell cultures with various mutations in the MAPK pathway. Proliferative melanoma cells were more susceptible to pathway inhibition than invasive phenotype cells, irrespective of BRAF mutation status, while invasive phenotype cell response was dependent on BRAF mutation status. Critically, MAPK pathway inhibition of proliferative phenotype cells resulted in acquisition of invasive phenotype characteristics. These results show that melanoma cell phenotype is an important factor in MAPK pathway inhibition response. This suggests that while current therapeutic strategies target proliferative melanoma cells, future approaches should also account for the invasive phenotype population.
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.
Mycosis fungoide (MF) patients who develop tumors or extracutaneous involvement usually have a poor prognosis with no curative therapy available so far. In the present European Organization for ...Research and Treatment of Cancer (EORTC) multicenter study, the genomic profile of 41 skin biopsies from tumor stage MF (MFt) was analyzed using a high-resolution oligo-array comparative genomic hybridization platform. Seventy-six percent of cases showed genomic aberrations. The most common imbalances were gains of 7q33.3q35 followed by 17q21.1, 8q24.21, 9q34qter, and 10p14 and losses of 9p21.3 followed by 9q31.2, 17p13.1, 13q14.11, 6q21.3, 10p11.22, 16q23.2, and 16q24.3. Three specific chromosomal regions, 9p21.3, 8q24.21, and 10q26qter, were defined as prognostic markers showing a significant correlation with overall survival (OS) (P=0.042, 0.017, and 0.022, respectively). Moreover, we have established two MFt genomic subgroups distinguishing a stable group (0–5 DNA aberrations) and an unstable group (>5 DNA aberrations), showing that the genomic unstable group had a shorter OS (P=0.05). We therefore conclude that specific chromosomal abnormalities, such as gains of 8q24.21 (MYC) and losses of 9p21.3 (CDKN2A, CDKN2B, and MTAP) and 10q26qter (MGMT and EBF3) may have an important role in prognosis. In addition, we describe the MFt genomic instability profile, which, to our knowledge, has not been reported earlier.
To date there is no effective therapy for metastatic melanoma and at the molecular level the disease progression is poorly understood. A recent study by our group led to the development of a novel ...phenotype switching model for melanoma progression, wherein cells transition back-and-forth between states of proliferation and invasion to drive disease progression. To explore the model's clinical relevance we interrogated phenotype-specific expression patterns in human melanoma patient material. A matched primary/metastasis pair from a human melanoma patient was obtained and immunohistochemically stained for proliferative and invasive phenotype markers. These were also stained for hypoxia and blood vessel markers. Proliferative phenotype markers Melan-A and Mitf showed consistent anti-correlation with invasive phenotype marker Wnt5A and hypoxia marker Glut-1. These also correlated with observed intra-tumoural vascularization patterns. Similar pattern distributions were present in both primary and metastasis samples. Strikingly, we observed that late phase metastatic melanoma cells adopt morphologies and behaviours identical to very early phase cells. The expression patterns observed closely matched expectations derived from previous in vitro and xenografting experiments. These results highlight the likelihood that disease progression involves melanoma cells retaining the capacity to regulate the expression of metastatic potential critical factors according to changing microenvironmental conditions.
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.
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