[Current Therapies in Superficial Malignant Tumors]. / Aktuelle Therapiekonzepte bei oberflächlichen malignen Tumoren.

This article is a review of diagnostic and therapeutic possibilities in common epibulbar malignant tumors, such as basal cell carcinoma, conjunctival lymphoma, squamous cell carcinoma and conjunctival melanoma. Most importantly, for every tumor patient there is a detailed anamnesis, split lamp examination and photo documentation. Further regular controls after therapy are required due to the risk of recurrences. Basal cell carcinoma is the most common tumor of the periocular skin, divided into three subtypes (nodular, superficial and morphea). The treatment consists of total excision with a sufficient safety margin.The most common tumor of the orbital and ocular adnexa are lymphoma. They can appear primary or secondary, as lymphatic system disease. The gold standard therapy is a percutaneous irradiation.Squamous cell carcinoma and its precancerous stages (CIN I-III) are frequently diagnosed on the conjunctiva. A complete tumor excision is required, but sometimes it is not possible due to large tumor growth. Therefore, an adjuvant therapy with mitomycin C eyedrops, brachytherapy with ruthenium plaque or proton radiotherapy is required.Conjunctival melanoma is a rare, but life-threatening disease. Melanosis with atypia has a high risk for degeneration and requires further treatment with mitomycin C eyedrops. In cases of malignant tumor growth, an excision with a non-touch technique and adjuvant therapy with brachy or proton irradiation should be applied.

Proton radiotherapy in advanced malignant melanoma of the conjunctiva.

BACKGROUND: The management of conjunctival melanoma is challenging and frequently ends in exenteration. The aim of this retrospective study was to evaluate the long-term results of proton beam radiation with regard to various clinical parameters. METHODS: Eighty-nine patients with extended conjunctival melanoma (≥T2) and multifocal bulbar located tumors (T1c/d) were treated consecutively with proton radiotherapy (dose 45 Gy). The following parameters were assessed: TNM stage, tumor origin, local recurrence, performance of exenteration, occurrence of metastases, overall survival, and potential complications. A time-to-event analysis was preformed to the primary endpoints: relapse, metastasis, exenteration, and death by use of Kaplan-Meier cumulative survival estimates and Cox proportional hazards regression that provides hazard ratios and 95% confidence intervals. RESULTS: The median follow-up time was 4.2 years (max. 21.7 years). Local recurrence and metastatic disease occurred in 33% and 16% of patients, respectively. Exenteration-free survival and overall survival tended to be worse in T3 melanoma. No association between tumor origin and local recurrence, metastatic disease, or overall survival was observed. Main complications after proton radiotherapy were sicca-syndrome (30%), secondary glaucoma (11%), and limbal stem cell deficiency (8%). CONCLUSIONS: In summary, proton radiotherapy in conjunctival melanoma is an effective alternative to exenteration, with a 5-year cumulative probability of eye preservation of 69%.

[Current Therapies in Superficial Malignant Tumors]. / Aktuelle Therapiekonzepte bei oberflächlichen malignen Tumoren.

This article is a review of diagnostic and therapeutic possibilities in common epibulbar malignant tumors, such as basal cell carcinoma, conjunctival lymphoma, squamous cell carcinoma and conjunctival melanoma. Most importantly, for every tumor patient there is a detailed anamnesis, split lamp examination and photo documentation. Further regular controls after therapy are required due to the risk of recurrences.Basal cell carcinoma is the most common tumor of the periocular skin, divided into three subtypes (nodular, superficial and morphea). The treatment consists of total excision with a sufficient safety margin.The most common tumor of the orbital and ocular adnexa are lymphoma. They can appear primary or secondary, as lymphatic system disease. The gold standard therapy is a percutaneous irradiation.Squamous cell carcinoma and its precancerous stages (CIN I - III) are frequently diagnosed on the conjunctiva. A complete tumor excision is required, but sometimes it is not possible due to large tumor growth. Therefore, an adjuvant therapy with mitomycin C eyedrops, brachytherapy with ruthenium plaque or proton radiotherapy is required.Conjunctival melanoma is a rare, but life-threatening disease. Melanosis with atypia has a high risk for degeneration and requires further treatment with mitomycin C eyedrops. In cases of malignant tumor growth, an excision with a non-touch technique and adjuvant therapy with brachy or proton irradiation should be applied.

Lentiviral Vector Promoter is Decisive for Aberrant Transcript Formation.

Lentiviral vectors hold great promise for the genetic correction of various inherited diseases. However, lentiviral vector biology is still not completely understood and warrants the precise decoding of molecular mechanisms underlying integration and post-translational modification. This study investigated a series of self-inactivating (SIN) and full long terminal repeat (LTR) lentiviral vectors that contained different types of promoters with or without a transgene to gain deeper insights in lentiviral target site selection and potential perturbation of cellular gene expression. Using an optimized nonrestrictive linear amplification-mediated polymerase chain reaction (nrLAM-PCR) protocol, vector structure-dependent integration site profiles were observed upon transduction of mouse lin- hematopoietic progenitors in vitro. Initial target site selection mainly depended on the presence of the promoter while being independent of its nature. Despite the increased propensity for read-through transcription of SIN lentiviral vectors, the incidence of viral-cellular fusion transcript formation involving the canonical viral splice donor or cryptic splice sites was reduced in both unselected primary lin- cells and transformed 32D cells. Moreover, the strength of the internal promoter in vectors with SIN LTRs is decisive for in vitro selection and for the abundance of chimeric transcripts, which are decreased by moderately active promoters. These results will help to better understand vector biology and to optimize therapeutic vectors for future gene therapy applications.

Frequent GNAQ, GNA11, and EIF1AX Mutations in Iris Melanoma.

Purpose: The most common malignant intraocular tumors with a high mortality in adults are uveal melanomas. Uveal melanomas arise most frequently in the choroid or ciliary body (97%) and rarely in the iris (3%). Whereas conjunctival and posterior uveal (ciliary body and choroidal) melanomas have been studied in more detail genetically, little data exist regarding iris melanomas. Methods: In our study, we genetically analyzed 19 iris melanomas, 8 ciliary body melanomas, 3 ring melanomas, and 4 iris nevi. A targeted next-generation sequencing approach was applied, covering the mutational hotspot regions of nine genes known to be mutated in conjunctival and uveal melanoma (BRAF, NRAS, KIT, GNAQ, GNA11, CYSLTR2, SF3B1, EIF1AX, and BAP1). Results: Activating GNAQ or GNA11 hotspot mutations were detected in a mutually exclusive fashion in 84% (16/19) of iris melanomas. EIF1AX gene mutations also were frequent, detected in 42% (8/19) of iris melanomas. In 4 iris nevi, one GNAQ mutation was identified. GNAQ, GNA11, EIF1AX, and BAP1 mutations were identified at varying frequencies in ciliary body and ring melanomas. Conclusions: In this most comprehensive genetic analysis of iris melanomas published to date, we find iris melanomas to be related genetically to choroidal and ciliary body melanomas, frequently harboring GNAQ, GNA11, and EIF1AX mutations. Future studies will need to assess if screening mutation profiles in iris melanomas may be of diagnostic or prognostic value.

SF3B1 and BAP1 mutations in blue nevus-like melanoma.

Blue nevi are melanocytic tumors originating in the cutaneous dermis. Malignant tumors may arise in association with or resembling blue nevi, so called 'blue nevus-like melanoma', which can metastasize and result in patient death. Identifying which tumors will behave in a clinically aggressive manner can be challenging. Identifying genetic alterations in such tumors may assist in their diagnosis and prognostication. Blue nevi are known to be genetically related to uveal melanomas (eg, both harboring GNAQ and GNA11 mutations). In this study, we analyzed a large cohort (n=301) of various morphologic variants of blue nevi and related tumors including tumors diagnosed as atypical blue nevi (n=21), and blue nevus-like melanoma (n=12), screening for all gene mutations known to occur in uveal melanoma. Similar to published reports, we found the majority of blue nevi harbored activating mutations in GNAQ (53%) or GNA11 (15%). In addition, rare CYSLTR2 (1%) and PLCB4 (1%) mutations were identified. EIF1AX, SF3B1, and BAP1 mutations were also detected, with BAP1 and SF3B1 R625 mutations being present only in clearly malignant tumors (17% (n=2) and 25% (n=3) of blue nevus-like melanoma, respectively). In sequencing data from a larger cohort of cutaneous melanomas, this genetic profile was also identified in tumors not originally diagnosed as blue nevus-like melanoma. Our findings suggest that the genetic profile of coexistent GNAQ or GNA11 mutations with BAP1 or SF3B1 mutations can aid the histopathological diagnosis of blue nevus-like melanoma and distinguish blue nevus-like melanoma from conventional epidermal-derived melanomas. Future studies will need to further elucidate the prognostic implications and appropriate clinical management for patients with tumors harboring these mutation profiles.

Activating cysteinyl leukotriene receptor 2 (CYSLTR2) mutations in blue nevi.

Blue nevi are common melanocytic tumors arising in the dermal layer of the skin. Similar to uveal melanomas, blue nevi frequently harbor GNAQ and GNA11 mutations. Recently, recurrent CYSLTR2 and PLCB4 mutations were identified in uveal melanomas not harboring GNAQ or GNA11 mutations. All four genes (GNAQ, GNA11, CYSLTR2, and PLCB4) code for proteins involved in the same signaling pathway, which is activated by mutations in these genes. Given the related functional consequences of these mutations and the known genetic similarities between uveal melanoma and blue nevi, we analyzed a cohort of blue nevi to investigate whether CYSLTR2 and PLCB4 mutations occur in tumors lacking GNAQ or GNA11 mutations (as in uveal melanoma). A targeted next-generation sequencing assay covering known activating mutations in GNAQ, GNA11, CYSLTR2, PLCB4, KIT, NRAS, and BRAF was applied to 103 blue nevi. As previously reported, most blue nevi were found to harbor activating mutations in GNAQ (59%, n=61), followed by less frequent mutations in GNA11 (16%, n=17). Additionally, one BRAF (1%) and three NRAS (3%) mutations were detected. In three tumors (3%) harboring none of the aforementioned gene alterations, CYSLTR2 mutations were identified. All three CYSLTR2 mutations were the same c.386T>A, L129Q mutation previously identified in uveal melanoma that has been shown to lead to increased receptor activation and signaling. In summary, our study identifies CYSLTR2 L129Q alterations as a previously unrecognized activating mutation in blue nevi, occuring in a mutually exclusive fashion with known GNAQ and GNA11 mutations. Similar to GNAQ and GNA11 mutations, CYSLTR2 mutations, when present, are likely defining pathogenetic events in blue nevi.

Oncogene status as a diagnostic tool in ocular and cutaneous melanoma.

The majority of human tumours can be easily and correctly diagnosed based on clinical information and pathological assessment. In some cases however, correct diagnosis can prove difficult. In such cases, molecular approaches can be of significant diagnostic value. In recent years, the understanding of genetic alterations has greatly increased. In cutaneous melanoma, it is now well recognised, that 70-80% of tumours harbour BRAF and NRAS mutations. These mutations never occur in uveal melanoma. On the other hand activating GNAQ and GNA11 mutations are found in ∼90% of uveal melanomas, and are exceptionally rare in other melanomas (<1%). Here, we demonstrate a number of melanoma cases, where distinguishing if a tumour was of cutaneous or ocular origin was not possible based on clinical and pathological assessment. In these cases there was either atypical clinical presentation or metastasis of unclear primary. Histological distinction between uveal and cutaneous melanomas, especially at the stage of metastasis, is not reliable as they can be morphologically very similar. In all cases we present, a simple genetic assessment of oncogene mutation status was able to clearly define the melanoma type. This type of genetic assessment is of great diagnostic value and due to its simplicity could be performed in routine clinical practice even in smaller institutions.

Targeted next generation sequencing reveals unique mutation profile of primary melanocytic tumors of the central nervous system.

Melanocytic tumors originating in the central nervous system (MT-CNS) are rare tumors that generally have a favorable prognosis, however malignant tumors do occur. Pathogenetically MT-CNS are not well characterized. Similar to uveal melanoma and blue nevi, they frequently harbor activating GNAQ or GNA11 mutations. Rare NRAS mutations have also been reported. Other mutations have not yet been described. We analyzed 19 MT-CNS, 7 uveal melanomas and 19 cutaneous melanomas using a targeted next generation sequencing approach analyzing 29 genes known to be frequently mutated in other melanocytic tumors (in particular uveal and cutaneous melanomas). In concordance with previous studies, cutaneous melanoma samples showed frequent NRAS or BRAF mutations, as well as mutations in other genes (e.g. NF1, RAC1, PIK3CA, ARID1A). Metastasized uveal melanomas exhibited mutations in GNAQ, GNA11 and BAP1. In contrast, MT-CNS almost exclusively demonstrated mutations in GNAQ (71 %) or GNA11 (12 %). Interestingly both GNA11 mutations identified were detected in MT-CNS diagnosed as intermediate grade melanocytomas which also recurred. One of these recurrent cases also harbored an inactivating BAP1 mutation and was found to have lost one copy of chromosome 3. Our findings show that while MT-CNS do have GNAQ or GNA11 mutations, they rarely harbor other recurrent mutations found in uveal or cutaneous melanomas. Considering chromosome 3 and BAP1 loss are robust markers of poor prognosis in uveal melanoma, it will prove interesting to determine whether these genomic alterations are also of prognostic significance in MT-CNS.

Targeted massively parallel sequencing of angiosarcomas reveals frequent activation of the mitogen activated protein kinase pathway.

Angiosarcomas are rare malignant mesenchymal tumors of endothelial differentiation. The clinical behavior is usually aggressive and the prognosis for patients with advanced disease is poor with no effective therapies. The genetic bases of these tumors have been partially revealed in recent studies reporting genetic alterations such as amplifications of MYC (primarily in radiation-associated angiosarcomas), inactivating mutations in PTPRB and R707Q hotspot mutations of PLCG1. Here, we performed a comprehensive genomic analysis of 34 angiosarcomas using a clinically-approved, hybridization-based targeted next-generation sequencing assay for 341 well-established oncogenes and tumor suppressor genes. Over half of the angiosarcomas (n = 18, 53%) harbored genetic alterations affecting the MAPK pathway, involving mutations in KRAS, HRAS, NRAS, BRAF, MAPK1 and NF1, or amplifications in MAPK1/CRKL, CRAF or BRAF. The most frequently detected genetic aberrations were mutations in TP53 in 12 tumors(35%) and losses of CDKN2A in9 tumors (26%). MYC amplifications were generally mutually exclusive of TP53 alterations and CDKN2A loss and were identified in 8 tumors (24%), most of which (n = 7, 88%) arose post-irradiation. Previously reported mutations in PTPRB (n = 10, 29%) and one (3%) PLCG1 R707Q mutation were also identified. Our results demonstrate that angiosarcomas are a genetically heterogeneous group of tumors, harboring a wide range of genetic alterations. The high frequency of genetic events affecting the MAPK pathway suggests that targeted therapies inhibiting MAPK signaling may be promising therapeutic avenues in patients with advanced angiosarcomas.

Frequent TERT Promoter Mutations in Ocular Surface Squamous Neoplasia.

PURPOSE: Ocular surface squamous neoplasia, including intraepithelial neoplasia (CIN) and invasive squamous cell carcinoma (SCC), are one of the most common malignant tumors of the conjunctiva. Little is known of the genetic alterations involved in their pathogenesis. Promoter mutations in telomerase reverse transcriptase (TERT) have been identified in various cancers, including many associated with ultraviolet (UV) exposure. Our study analyzes the mutation rate and clinicopathological associations of TERT promoter mutations in ocular surface squamous neoplasia. METHODS: DNA was isolated and the region of the TERT promoter where hotspot mutations can occur analyzed by Sanger-sequencing in 48 ocular surface squamous neoplasia tumor samples (6 CIN and 42 SCC). An analysis of associations between TERT promoter mutation status and various clinicopathological parameters was performed. RESULTS: We identified TERT promoter mutations in 21 of 48 ocular surface squamous neoplasia samples (43.8%), including 4 in CIN and 17 in SCC. The mutations consisted of 8 Chr.5:1295228C>T, 1 Chr.5:1295228_1295229CC>TT, 5 Chr.5:1295242_1295243CC>TT, and 12 Chr.5:1295250C>T mutations. All mutations were C>T or CC>TT alterations, demonstrating a UV-signature. TERT promoter mutations showed no statistically significant associations with clinicopathological parameters. CONCLUSIONS: Telomerase reverse transcriptase promoter mutations are found in almost half of ocular surface squamous neoplasias and have a mutation profile supporting UV induction as the major source of mutagenesis. We conclude that UV induced TERT promoter mutations leading to aberrant overexpression of telomerase is a major pathogenetic factor in ocular surface squamous neoplasia.

Analysis of SDHD promoter mutations in various types of melanoma.

OBJECTIVES: Recently, recurrent mutations in regulatory DNA regions, such as promoter mutations in the TERT gene were identified in melanoma. Subsequently, Weinhold et al. reported SDHD promoter mutations occurring in 10% of melanomas and being associated with a lower overall survival rate. Our study analyzes the mutation rate and clinico-pathologic associations of SDHD promoter mutations in a large cohort of different melanoma subtypes. METHODS: 451 melanoma samples (incl. 223 non-acral cutaneous, 38 acral, 33 mucosal, 43 occult, 43 conjunctival and 51 uveal melanoma) were analyzed for the presence of SDHD promoter mutations by Sanger-sequencing. Statistical analysis was performed to screen for potential correlations of SDHD promoter mutation status with various clinico-pathologic criteria. RESULTS: The SDHD promoter was successfully sequenced in 451 tumor samples. ETS binding site changing SDHD promoter mutations were identified in 16 (4%) samples, of which 5 mutations had not been described previously. Additionally, 5 point mutations not located in ETS binding elements were identified. Mutations in UV-exposed tumors were frequently C>T. One germline C>A SDHD promoter mutation was identified. No statistically significant associations between SDHD promoter mutation status and various clinico-pathologic variables or overall patient survival were observed. CONCLUSIONS: Melanomas harbor recurrent SDHD promoter mutations, which occur primarily as C>T alterations in UV-exposed melanomas. In contrast to the initial report and promoter mutations in the TERT gene, our analysis suggests that SDHD promoter mutations are a relatively rare event in melanoma (4% of tumors) of unclear clinical and prognostic relevance.

High-throughput monitoring of integration site clonality in preclinical and clinical gene therapy studies.

Gene transfer to hematopoietic stem cells with integrating vectors not only allows sustained correction of monogenic diseases but also tracking of individual clones in vivo. Quantitative real-time PCR (qPCR) has been shown to be an accurate method to quantify individual stem cell clones, yet due to frequently limited amounts of target material (especially in clinical studies), it is not useful for large-scale analyses. To explore whether vector integration site (IS) recovery techniques may be suitable to describe clonal contributions if combined with next-generation sequencing techniques, we designed artificial ISs of different sizes which were mixed to simulate defined clonal situations in clinical settings. We subjected all mixes to either linear amplification-mediated PCR (LAM-PCR) or nonrestrictive LAM-PCR (nrLAM-PCR), both combined with 454 sequencing. We showed that nrLAM-PCR/454-detected clonality allows estimating qPCR-detected clonality in vitro. We then followed the kinetics of two clones detected in a patient enrolled in a clinical gene therapy trial using both, nrLAM-PCR/454 and qPCR and also saw nrLAM-PCR/454 to correlate to qPCR-measured clonal contributions. The method presented here displays a feasible high-throughput strategy to monitor clonality in clinical gene therapy trials is at hand.

From bench to bedside: preclinical evaluation of a self-inactivating gammaretroviral vector for the gene therapy of X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by impaired antimicrobial activity in phagocytic cells. As a monogenic disease affecting the hematopoietic system, CGD is amenable to gene therapy. Indeed in a phase I/II clinical trial, we demonstrated a transient resolution of bacterial and fungal infections. However, the therapeutic benefit was compromised by the occurrence of clonal dominance and malignant transformation demanding alternative vectors with equal efficacy but safety-improved features. In this work we have developed and tested a self-inactivating (SIN) gammaretroviral vector (SINfes.gp91s) containing a codon-optimized transgene (gp91(phox)) under the transcriptional control of a myeloid promoter for the gene therapy of the X-linked form of CGD (X-CGD). Gene-corrected cells protected X-CGD mice from Aspergillus fumigatus challenge at low vector copy numbers. Moreover, the SINfes.gp91s vector generates substantial amounts of superoxide in human cells transplanted into immunodeficient mice. In vitro genotoxicity assays and longitudinal high-throughput integration site analysis in transplanted mice comprising primary and secondary animals for 11 months revealed a safe integration site profile with no signs of clonal dominance.

The ß-globin locus control region in combination with the EF1α short promoter allows enhanced lentiviral vector-mediated erythroid gene expression with conserved multilineage activity.

Some gene therapy strategies are compromised by the levels of gene expression required for therapeutic benefit, and also by the breadth of cell types that require correction. We designed a lentiviral vector system in which a transgene is under the transcriptional control of the short form of constitutively acting elongation factor 1α promoter (EFS) combined with essential elements of the locus control region of the ß-globin gene (ß-LCR). We show that the ß-LCR can upregulate EFS activity specifically in erythroid cells but does not alter EFS activity in myeloid or lymphoid cells. Experiments using the green fluorescent protein (GFP) reporter or the human adenosine deaminase (ADA) gene demonstrate 3-7 times upregulation in vitro but >20 times erythroid-specific upregulation in vivo, the effects of which were sustained for 1 year. The addition of the ß-LCR did not alter the mutagenic potential of the vector in in vitro mutagenesis (IM) assays although microarray analysis showed that the ß-LCR upregulates ~9% of neighboring genes. This vector design therefore combines the benefits of multilineage gene expression with high-level erythroid expression, and has considerable potential for correction of multisystem diseases including certain lysosomal storage diseases through a hematopoietic stem cell (HSC) gene therapy approach.

Genome-wide high-throughput integrome analyses by nrLAM-PCR and next-generation sequencing.

High-throughput integration site profiling has become a feasible tool to assess vector biosafety and to monitor the cell fate of the gene-corrected cell population in clinical gene therapy studies. Here we report a step-by-step protocol for universal genome-wide and comprehensive integrome analysis that can be performed on >10(2)-10(3) samples of interest in parallel. This assay is composed of fast and cost-efficient non-restrictive linear amplification-mediated PCR; optimized sample preparation for pyrosequencing; and automated bioinformatic data mining, including sequence trimming, alignment to the cellular genome and further annotation. Moreover, the workflow of this large-scale assay can be adapted to any PCR-based method aiming to characterize unknown flanking DNA adjacent to a known DNA region. Thus, in combination with next-generation sequencing technologies, large-scale integrome analysis of > 4 x 10(5)-1 x 10(6) integration site sequences can be accomplished within a single week.

A Ubc7p-binding domain in Cue1p activates ER-associated protein degradation.

Cue1p is an N-terminally anchored endoplasmic reticulum (ER) protein essential for the activity of the two major yeast RING finger ubiquitin ligases (E3s) implicated in ER-associated degradation (ERAD). Cue1p contains a CUE domain, which for several proteins is known to bind ubiquitin. We now establish that the CUE domain is dispensable for ERAD of substrates of both Hrd1p and Doa10p and that the Cue1p transmembrane domain is similarly not required for degradation of the Hrd1p substrate CPY. Cue1p interacts with the ERAD E2 Ubc7p in vivo. We show that a discrete C-terminal Ubc7p binding region (U7BR) of Cue1p is required for ERAD and for Ubc7p-dependent ubiquitylation by Hrd1p in vitro. Strikingly, when Ubc7p is stabilized by direct anchoring to the ER membrane, the U7BR is sufficient to restore ERAD in cells lacking Cue1p. Thus, discrete E2 binding sites independent of ubiquitin ligase domains have the potential to activate ubiquitylation.