Maria B. Karpova

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 α-helix C and showed robust (≈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.

Oligonucleotide Array-CGH Identifies Genomic Subgroups and Prognostic Markers for Tumor Stage Mycosis Fungoides

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.

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.

Sorafenib in melanoma.

Introduction: Sorafenib is an orally available multi-kinase inhibitor that inhibits tumor proliferation by targeting multiple kinases including the vascular endothelial growth factor receptors VEGFR1, VEGFR2, VEGFR3 and the platelet-derived growth factor receptor PDGFR, and it targets tumor progression by inhibiting FLT3, C-Kit and BRAF. Since BRAF mutations are frequent in melanoma, sorafenib was investigated in various Phase I, II and III clinical trials. The drug is well tolerated with mild to moderate adverse effects, which are mostly limited to cutaneous toxicity, diarrhea and fatigue. Areas covered: Systematic literature review of the randomized trials using PubMed was performed. Original articles were reviewed and citations from those were also considered. Additionally, clinical trial databases were examined to identify and summarize ongoing trials of sorafenib in melanoma patients. Expert opinion: Sorafenib as a monotherapy or in combination with chemotherapy is of limited use. Combining it with dacarbazine doubled the response rate and the progression-free survival in metastatic melanoma patients. Unfortunately, these results have never been evaluated in large randomized Phase III clinical trials. According to the trials conducted so far a subpopulation of patients experience substantial benefit, therefore it is essential to identify biomarkers to select the subgroups of patients that are more likely to respond to sorafenib. Furthermore, other less frequent subtypes such as mucosal or ocular melanoma still constitute promising targets; academic institutions are currently launching investigator-initiated trials in these indications.

Primary cutaneous lymphoma: Two-decade comparison in a population of 263 cases from a Swiss tertiary referral centre

Background  Epidemiological data on primary cutaneous lymphomas (PCLs) are rare and have not previously been investigated in Switzerland.

Evaluation of lymphangiogenic markers in Sézary syndrome.

Sézary syndrome (SS) is regarded as a leukemic, aggressive subtype of cutaneous T-cell lymphoma (CTCL) characterized by the accumulation of malignant T-cells in the skin, as well as by blood and lymph node involvement. To date there have been no data on the extent of lymphangiogenesis in SS or erythrodermic mycosis fungoides (eMF). Lymphangiogenesis represents the de novo formation of lymphatic vasculature and has been associated with the occurrence of metastatic disease and poor prognosis. In this study we investigated lymphangiogenesis in skin biopsies from patients with SS and eMF. The expression of VEGFR-3 was significantly higher in patients with SS (p = 0.0285) as compared to patients with eMF. LYVE-1, podoplanin (PDPN), and VEGF-C stainings showed a similar tendency. The number of PDPN-expressing lymphatic vessels (p = 0.025) as well as CD31-positive blood vessels (p = 0.0065) correlated with disease progression in patients with SS. We show for the first time a non-vascular pattern of VEGF-C and VEGFR-3, i.e. their epidermal expression in erythrodermic CTCLs, suggesting their role in lymphocyte trafficking to the skin.

Complete absence of the αGal xenoantigen and isoglobotrihexosylceramide in α1,3galactosyltransferase knock‐out pigs

Puga Yung GL, Li Y, Borsig L, Millard A‐L, Karpova MB, Zhou D, Seebach JD. Complete absence of the αGal xenoantigen and isoglobotrihexosylceramide in α1,3galactosyltransferase knock‐out pigs. Xenotransplantation 2012; 19: 196–206.

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-Hodgkins 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.

Primary Cutaneous CD30+ Anaplastic Large-Cell Lymphomas Show a Heterogeneous Genomic Profile: An Oligonucleotide ArrayCGH Approach

TO THE EDITOR Primary cutaneous CD30-positive lymphoproliferative disorders are the second most common group of primary cutaneous T-cell lymphomas (Willemze et al., 2005), after the mycosis fungoides/Sézary syndrome group. The clinical and histological features of primary cutaneous anaplastic large-cell lymphoma (C-ALCL) have been well characterized, but little is known about its underlying pathogenetic and genetic alterations. Previous comparative genomic hybridization (CGH) studies focusing on C-ALCL that included a limited number of samples yielded nonhomogeneous results (Böni et al., 2000; Mao et al., 2003; Prochazkova et al., 2003; Fischer et al., 2004; Zettl et al., 2004). Recently, three studies that were based on array CGH (aCGH) and included a small number of C-ALCL patients were published (Mao et al., 2003; Laharanne et al., 2010; van Kester et al., 2010). We have investigated the genomic profile of 19 C-ALCL patients using a 60-mer 44K oligonucleotide-arrayCGH platform and compared our results with those of previous aCGH studies. C-ALCL patients were selected according to the World Health Organization–European Organization for Research and Treatment for Cancer (EORTC) classification for cutaneous lymphomas (Willemze et al., 2005). This EORTC multicenter study was conducted in the departments of pathology and dermatology of six European centers in Spain and Switzerland. The local ethics committees approved the study, and written informed consent was obtained from all patients, in accordance the Declaration of Helsinki Principles. Clinical characteristics are detailed in Supplementary Table S1 online. The study was performed with 20 10 mm snap-frozen C-ALCL samples to ensure the high quality of the DNA. Hematoxylin–eosin staining of a frozen section of each sample was performed tumor cell infiltration of at least 70%. DNA was isolated using a commercial kit as described in manufacturer’s instructions (Dneasy Blood and Tissue Kit; Qiagen, Hilden, Germany). Genomewide analysis of patient samples was conducted using the Human Genome CGH 44K microarrays (G4410B and G4426B; Agilent Technologies, Palo Alto, CA), a whole-genome platform containing 44,000 probes along the entire human genome with a mean resolution of ±75 kb. Hybridization was performed according to the manufacturer’s protocols. Data analysis was conducted as previously described (Salgado et al., 2010). Fluorescence in situ hybridization with noncommercial probes of bacterial artificial chromosome DNA clones from the CHORI bacterial artificial chromosome/PAC resource (http://bacpac. chori.org) was performed to confirm chromosomal abnormalities previously detected by aCGH in cases for which a paraffin-embedded tissue biopsy was available. Chromosomal abnormalities were detected in 17 of 19 analyzed C-ALCL samples (89.5%). Losses were more frequently detected than gains (78.9 vs. 68.4%). Mao et al. (2003) and van Kester et al. (2010) found gains more frequently than losses, whereas Laharanne et al. (2010) detected losses more frequently. The highest frequencies of chromosomal aberrations were 60% (Mao et al., 2003) and 45% (Laharanne et al., 2010; van Kester et al., 2010), in contrast to 36.8% in our present study. Regarding the smallest overlapping regions of imbalance, 15 corresponded to losses and 9 to gains. The results are summarized in Figure 1 and detailed in Supplementary Table S2 online. The specific chromosomal regions and candidate genes mapped in these regions are detailed in Table 1. The most frequent abnormalities observed were deletions located on 16q, 13q, 17p13, and 20q13. Genomic losses of 13q34 (ING1) and 16q22.11 (CTCF) detected by aCGH were confirmed by fluorescence in situ hybridization in three patients. No significant correlation between the observed clinical features and the presence of chromosomal aberrations could be demonstrated. Furthermore, no data regarding the prognostic significance of the observed genetic results were obtained. In agreement with studies by van Kester et al. (2010) and Laharanne et al. (2010), two regions were lost in our study, at 13q33.3 and 16p11.2. These regions were not detected in the first aCGH study (Mao et al., 2003), probably because they may not have been among the 57 oncogenic regions of the AmpliOnc platform. Similar to the findings of van Kester et al. (2010), we observed losses at 3p26.3, 6q21, 8p22, 13q12.11, 13q13.1, 16p11.2-16q11.2, 17p13.1, and 17p13.3 (Supplementary Table S3 online). The main concordance between our results and those of van Kester et al. (2010) was a deletion at 16q11.2. However, differences were observed for a higher frequency of 16q losses in our series, including seven genomic regions located between 16q11.2 and 16q24.3. The most Abbreviations: aCGH, array comparative genomic hybridization; C-ALCL, primary cutaneous anaplastic large-cell lymphoma; CGH, comparative genomic hybridization

Split-face study of melasma patients treated with non-ablative fractionated photothermolysis (1540 nm)

Background  Melasma treatment remains challenging despite various laser systems available, because of potential side‐effects and high recurrence rates.