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.

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

BACKGROUND: Anti-Galα1,3Galß-R natural antibodies are responsible for hyperacute rejection in pig-to-primate xenotransplantation. Although the generation of pigs lacking the α1,3galactosyltransferase (GalT) has overcome hyperacute rejection, antibody-mediated rejection is still a problem. It is possible that other enzymes synthesize antigens similar to Galα1,3Gal epitopes that are recognized by xenoreactive antibodies. The glycosphingolipid isoglobotrihexosylceramide (iGb3) represents such a candidate expressing an alternative Galα1,3Gal epitope. The present work determined whether the terminal Galα1,3Gal disaccharide is completely absent in Immerge pigs lacking the GalT using several different highly sensitive methods. METHODS: The expression of Galα1,3Gal was evaluated using a panel of antibodies and lectins by flow cytometry and fluorescent microscopy; GalT activity was detected by an enzymatic assay; and ion trap mass spectroscopy of neutral cellular membranes extracted from aortic endothelial was used for the detection of sugar structures. Finally, the presence of iGb3 synthase mRNA was tested by RT-PCR in pig thymus, spleen, lymph node, kidney, lung, and liver tissue samples. RESULTS: Aortic endothelial cells derived from GalT knockout pigs expressed neither Galα1,3Gal nor iGb3 on their surface, and GalT enzymatic activity was also absent. Lectin staining showed an increase in the blood group H-type sugar structures present in GalT knockout cells as compared to wild-type pig aortic endothelial cells (PAEC). Mass spectroscopic analysis did not reveal Galα1,3Gal in membranes of GalT knockout PAEC; iGb3 was also totally absent, whereas a fucosylated form of iGb3 was detected at low levels in both pig aortic endothelial cell extracts. Isoglobotrihexosylceramide 3 synthase mRNA was expressed in all pig tissues tested whether derived from wild-type or GalT knockout animals. CONCLUSIONS: These results confirm unequivocally the absence of terminal Galα1,3Gal disaccharides in GalT knockout endothelial cells. Future work will have to focus on other mechanisms responsible for xenograft rejection, in particular non-Galα1,3Gal antibodies and cellular responses.

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.

Combined spectral karyotyping, comparative genomic hybridization, and in vitro apoptyping of a panel of Burkitt's lymphoma-derived B cell lines reveals an unexpected complexity of chromosomal aberrations and a recurrence of specific abnormalities in chemoresistant cell lines.

The comprehensive cytogenetic profiles of a panel of 10 Burkitt's lymphoma (BL)-derived B cell lines, designated Akata, BL-28, BL-41, Daudi, DG-75, Mutu I, Mutu III, Namalwa, Rael, and Ramos, respectively, are reported herein. The unique origin of each cell line was established using multiplex quantitative fluorescence polymerase chain reaction (QF-PCR). Spectral karyotyping (SKY) revealed a large number of structural and numerical chromosomal aberrations, many of which had not been previously identified or resolved by conventional G-banding techniques. Notably, whereas all 10 cell lines harbored the hallmark translocation t(8;14)(q24;q32), no other common structural aberrations were identified, although translocations involving chromosomes 3, 13, and 17 were frequently seen. Moreover, analysis of chromosomal breakpoints by comparative genomic hybridization (CGH) revealed a number of recurring aberrations, such as gain of chromosomes 7 and 20, gains of regions at 2p, 3q, 13q and 16q, and losses at 3p, 4q and 17p. In addition, apoptyping (i.e. determination of in vitro responses to apoptosis stimulation) of the cell lines suggested specific association patterns between karyotypic changes (e.g. translocations involving 17p, and gains of portions of chromosomes 7 and 20) and resistance to the chemotherapeutic agent, etoposide. The current molecular cytogenetic characterization of 10 BL cell lines has thus identified several novel sites of rearrangements; moreover, the combined karyotyping and functional assessment (apoptyping) of these cell lines serves to enhance their utility in future studies aimed at gene discovery and gene function.

Combined CYP1A1/GSTM1 at-risk genotypes are overrepresented in squamous cell lung carcinoma patients but underrepresented in elderly tumor-free subjects.

PURPOSE: Polycyclic aromatic hydrocarbons are activated by cytochrome P450 1A1 (CYP1A1) and inactivated by glutathione S-transferase mu (GSTM1). Therefore, it is expected that a combination of proficient CYP1A1 genotype with deficient GSTM1 variant would result in particularly elevated lung cancer (LC) risk, especially for squamous cell carcinoma (SCC). This study was aimed to validate whether the CYP1A1-C (3801) (CYP1A1*2) allele has an unfavorable significance alone and/or in combination with the GSTM1 deficiency. METHODS: We compared the distribution of CYP1A1 and GSTM1 genotypes in LC patients (n=141), healthy donors (HD, n=204), and elderly tumor-free smokers and non-smokers (ED, n=246). RESULTS: CYP1A1*2 allele carriers demonstrated a clear-cut association with SCC: the adjusted odds ratios (OR) were 2.22 (95% CI=1.06-4.63) and 2.27 (95% CI=1.14-4.52) when HD and ED were used as referents, respectively. CYP1A1*2(+)/GSTM1(-) combined genotypes were overrepresented in the SCC patients (14/70, 20.0%) and underrepresented in the ED (19/246, 7.7%) as compared to the intermediate prevalence in the HD (26/204, 12.7%); the adjusted OR for SCC versus ED reached 3.85 (95% CI=1.43-10.33). CONCLUSIONS: In agreement with some literature data, our results support the concerted role of CYP1A1 and GSTM1 at-risk genotypes in SCC predisposition.

'Comparison of extremes' approach provides evidence against the modifying role of NAT2 polymorphism in lung cancer susceptibility.

NAT2 (arylamine N-acetyltransferase 2) polymorphism, being a key determinant of individual variations in acetylation capacity, is suspected to modify the risk of carcinogen-related malignancies. As tobacco smoke and other inhaled hazards contain a variety of NAT2 substrates, the relationship between NAT2 phenotype and lung cancer (LC) risk has been a subject of intensive research, however different case-control studies produced controversial data. In the present report, we employed a novel 'comparison of extremes' approach, i.e. we compared the distribution of NAT2 genotypes in lung cancer patients (LC, n=178) not only to the population controls (healthy donors (HD), n=364), but also to the subjects with a putative cancer-resistant constitution (elderly tumor-free smokers and non-smokers (ED), n=351). Frequencies of homozygous rapid, heterozygous rapid and slow acetylators were 6, 39 and 56% in LC, 8, 32 and 60% in HD, and 6, 35 and 59% in ED, respectively. Comparison of the NAT2 genotype frequencies between affected and non-affected individuals did not reveal any statistical deviations, irrespectively of smoking history, gender, age, or histological type of LC. Adjusted odds ratio for rapid vs. slow acetylators was 1.12 (95% confidence intervals (CI): 0.73-1.74) comparing LC vs. HD, and 1.10 (95% CI: 0.74-1.62) comparing LC vs. ED. Similar distribution of NAT2 acetylator genotypes both in tumor-prone and in tumor-resistant groups suggests that, despite the presence of NAT2 carcinogenic substrates in tobacco smoke, NAT2 polymorphism does not play a noticeable role in lung cancer susceptibility.

In vivo profiling reveals immunomodulatory effects of sorafenib and dacarbazine on melanoma.

Sorafenib is a multi-kinase inhibitor used alone or in combination with dacarbazine to treat metastasized melanoma. Our study investigated the relationship between metabolic response assessed by PET-CT and global transcriptome changes during sorafenib and dacarbazine therapy in patients with advanced melanoma. We conducted an open-label, investigator-initiated study that enrolled 13 sorafenib-naïve Stage IV melanoma patients, whose metastases were accessible for repeated biopsies. Treatment regimen included orally administered sorafenib and intravenous dacarbazine. Biopsies of skin or superficial lymph node metastases were taken before treatment (baseline), during sorafenib and after dacarbazine therapy and used for transcriptional profiling and validation experiments. Serum samples were evaluated for cytokine production. Metabolic response to therapy was observed in 45.5% of patients. The study drugs were well tolerated. We observed a clear upregulation of interferon (IFN)-stimulated immune response genes in profiled metastases. The IFNγ-induced gene signature seemed to be enhanced after addition of dacarbazine to sorafenib. Serum IFNγ also increased during therapy, particularly after addition of dacarbazine. Induction of IFNγ stimulated genes correlating with increased serum IFNγ was predictive of better clinical outcome and responders who had significantly higher serum IFNγ levels lived longer. Our data reveal in situ changes in melanoma metastases during treatment with sorafenib and dacarbazine and suggest an additional mechanism of action through immunomodulation.

CYP17 genetic polymorphism in endometrial cancer: are only steroids involved?

Initiation and/or promotion of endometrial cancer is known to be associated with estrogen and androgen (androstenedione) excess as well as with hyperinsulinemia/insulin resistance. It is possible that some allelic polymorphisms of the genes involved in steroidogenesis or steroid metabolism contribute to endometrial cancer susceptibility. We evaluated here the role of CYP17 biallelic (MspAI) polymorphism in 114 endometrial cancer patients compared with 182 healthy women. Our data demonstrated that A2/A2 CYP17 genotype, considered on the basis of initial breast cancer studies as 'unfavorable', was under-represented in endometrial cancer group (odds ratio 0.48, 95% confidence interval 0.25-0.89) that confirmed results of two other recent investigations. Carriers of this genotype were characterized by having lower blood insulin (by 120 min of oral glucose tolerance test 36.7+/-3.9 microU/ml vs. 90.4+/-16.7 microU/ml in postmenopausal women with A1/A1 genotype, P=0.04) and C-peptide levels (after night fasting 575.2+/-78.3 pg/ml vs. 978.9+/-115.7 pg/ml, respectively, P=0.04). No significant difference was found between the mean concentrations of testosterone, dehydroepiandrosterone sulfate and estradiol concentrations in patients-carriers of separate CYP17 genotypes. Thus, CYP17 polymorphism (namely, carrying the 'normal' A1/A1 genotype) might be one of the risk factors for endometrial cancer development. A1/A1 CYP17 variant may be associated with untraditional (non-steroidal) pathways that calls for corresponding preventive measures in high-risk groups.

A novel approach for assessment of cancer predisposing roles of GSTM1 and GSTT1 genes: use of putatively cancer resistant elderly tumor-free smokers as the referents.

We applied an alternative approach to assess the controversial evidence for the role of GSTM1 and GSTT1 deficiencies (null genotypes) in cancer susceptibility. In this study setting, the prevalence of GSTM1 and GSTT1 null genotypes in the lung cancer patients (LCs, n = 167) were compared with those in the group of putatively cancer resistant individuals, i.e. elderly tumor-free donors (EDs, n = 324). Healthy middle-aged donors (HDs, n = 339) were used as another comparison group. Our results support the previous conclusions of a modest protective effect associated with presence of at least one functional copy of GSTM1 gene; the prevalence of GSTM1 deficiency in LCs (54%) did not differ from that observed in HDs (54%), but showed a significant increase when compared with EDs (45%) (OR = 1.46, 95% CI = 1.00-2.12). Furthermore, in agreement with mechanistic considerations, the GSTM1 null genotypes were more prevalent in squamous cell carcinoma patients (58%) and in lung cancer patients with seemingly low cumulative carcinogen exposure dose (non-smokers: 63%; patients aged below 50 years: 76%). Contrary to GSTM1, no significant effect in the lung cancer proneness was observed for the GSTT1 genotypes. The results of this study are thus in good agreement with the body of literature data, including several published meta-analyses. Consequently, the suggested study design involving additional "cancer resistant" group of non-affected subjects appears to provide highly demonstrative data and to be well suited for pilot investigations and for resolving controversial issues.

Coexpression of SOX10/CD271 (p75(NTR)) and ß-Galactosidase in Large to Giant Congenital Melanocytic Nevi of Pediatric Patients.

BACKGROUND: Congenital melanocytic nevi (CMNs) are melanocytic neoplasms that can transform into melanoma. However, this development is impeded in the majority of cases and mostly affects patients with large or giant CMNs. METHODS: To elucidate mechanisms that keep CMNs from malignant transformation, CMN tissue biopsies were investigated for p-ERK and senescence markers by immunohistochemistry and for SOX10/CD271 (p75(NTR)) by immunofluorescence. CMN cells were cultivated, and MTT assays were performed for evaluating cell viability. Mutation status for NRAS and BRAF was performed by real-time PCR. RESULTS: 13 CMNs (from patients aged 0.5-11.8 years, mean: 2.7) showed immunoreactivity for SOX10/CD271 (p75(NTR)) in 34.2%. p-ERK was immunoreactive in 80% (4/5); ß-galactosidase was significantly stronger expressed in CMNs compared to melanocytic nevi of patients over 70 years (p = 0.0085). The 5 CMN cultures were immunoreactive for SOX10/CD271 (p75(NTR)) in 36.7%. By silencing SOX10 by siRNA in 2 CMN cell cultures, cell viability decreased significantly. NRAS(Q61K) mutation was found in 91.7% (11/12) and BRAF(V600E) in 6.3% of all analyzable CMNs (1/16). CONCLUSIONS: Oncogene-induced senescence might prevent malignant transformation through activation of the mitogen-activated protein kinase pathway. SOX10 is necessary for the viability of human CMN cell cultures and may be responsible for clinical changes during aging.

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.

Cutaneous lymphomas: molecular pathways leading to new drugs.

Currently, cutaneous lymphomas represent a paradigm for the heterogeneity and the dynamic variability of neoplastic disorders resulting in the accumulation of clonal lymphocytes in the skin, and thus mirror the complexity of lymphocytic populations. Increasing knowledge and insight in pathobiology offer new opportunities for targeted interventions to selectively hit the tumor populations. mAbs, rexinoids, small kinase inhibitors, or molecules interfering with methylation or histone acetylation contribute to disease control. The rational and well-coordinated application of these tools, together with improved chemotherapeutic options, will hopefully further improve treatment success in the near future.

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.

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.

Betulinic acid, a natural cytotoxic agent, fails to trigger apoptosis in human Burkitt's lymphoma-derived B-cell lines.

Betulinic acid (BA), a pentacyclic triterpene of natural origin, effectively induces apoptosis in neuroectodermal tumors and was recently shown to be a potent trigger of cell death in human leukemia-derived cell lines. To explore the potential of BA in the treatment of hematologic malignancies, we tested a panel of 10 Burkitt's lymphoma (BL)-derived B-cell lines for sensitivity to BA. The human Jurkat T leukemia cell line was included as a positive control. Our studies show that BA exerts cytotoxic effects in some of the BL cell lines tested, including DG75, a chemoresistant BL cell line. However, cell death was caspase-independent, as evidenced by a lack of protection by zVAD-fmk, a pancaspase inhibitor, and displayed signs of necrosis. Furthermore, BA-induced caspase activation was seen to a minor extent in only 1 of the 10 BL cell lines tested (Ramos, a p53-deficient cell line), but was readily detected in Jurkat cells. Together, these studies indicate that resistance to BA-induced apoptosis is a common feature of BL-derived cell lines.