Constitutive activation of Gαq signaling by mutations in GNAQ or GNA11 occurs in over 80% of uveal melanomas (UMs) and activates MAPK. Protein kinase C (PKC) has been implicated as a link, but the ...mechanistic details remained unclear. We identified PKC δ and ɛ as required and sufficient to activate MAPK in GNAQ mutant melanomas. MAPK activation depends on Ras and is caused by RasGRP3, which is significantly and selectively overexpressed in response to GNAQ/11 mutation in UM. RasGRP3 activation occurs via PKC δ- and ɛ-dependent phosphorylation and PKC-independent, DAG-mediated membrane recruitment, possibly explaining the limited effect of PKC inhibitors to durably suppress MAPK in UM. The findings nominate RasGRP3 as a therapeutic target for cancers driven by oncogenic GNAQ/11.
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•PKC δ/ɛ mediate ERK activation in uveal melanoma with Gαq pathway mutations•The RAS exchange factor RasGRP3 is a critical module for ERK activation•PKC δ, PKC ɛ, and RasGRP3 are novel therapeutic targets for uveal melanoma
Chen et al. find that Ras is required for GNAQ-mediated MAPK activation and identify PKC δ,ɛ and RasGRP3 as components of a signaling module necessary and sufficient to activate the Ras/MAPK pathway in GNAQ mutant uveal melanoma (UM). RasGRP3 is selectively overexpressed in response to GNAQ/11 mutations in UM.
Ocular melanoma is a rare but often deadly malignancy that arises in the uvea (commonest primary site), conjunctiva or the orbit. Primary orbital melanoma (POM) is exceedingly rare, with ...approximately 60 cases reported to date. Despite recent advances in our understanding of the genetics of primary uveal and conjunctival melanomas, this information is lacking for POM.
DNA was extracted from 12 POM tissues, with matched germline DNA (where available). MLPA was conducted to detect chromosomal alterations and Sanger sequencing used to identify point mutations in candidate melanoma driver genes (BRAF, NRAS, KRAS, GNA11, GNAQ), and other genes implicated in melanoma prognosis (EIF1AX, SF3B1). Immunohistochemistry was performed to analyse BAP1 nuclear expression.
MLPA detected copy number alterations in chromosomes 1p, 3, 6 and 8. Sequencing of melanoma driver genes revealed GNAQ (p.Q209L) mutations in two samples; although it is possible that these samples represent extraocular spread of an occult uveal melanoma. A recurrent mutation in SF3B1 (p.R625H) was observed in indolent, but not aggressive, tumours; a mutation in EIF1AX (p.N4S) was detected in one patient with non-aggressive disease.
EIF1AX and SF3B1 mutations appear have a role in determining the clinical course of POM and detection of these changes could have clinical significance. Further in depth analysis of this rare group using differing 'omic technologies will provide novel insights into tumour pathogenesis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
To develop parsimonious models for estimating metastasis mortality in patients with choroidal melanoma for situations where use of the Liverpool Uveal Melanoma Prognosticator Online (LUMPO) or Tumor, ...Node, Metastasis (TNM) staging system is not possible.
A backward-selection algorithm identified largest basal tumor diameter (LBTD) and chromosome 3 status (C3S) as the most informative predictors of metastatic death. We defined two prognostic models, based on LBTD with or without known C3S, that took into account competing risks of death from other causes by using the Aalen estimator. The bootstrap procedure was used to estimate discrimination accuracy, expressed by the C-index.
The cohort was comprised of 8348 patients with choroidal melanoma, 4174 of whom had known chromosome 3 status; of the 1553 deaths that occurred among these patients, 956 were attributed to metastasis. For LBTD with or without known C3S, the metastatic-death-specific C-indices at 2, 5, and 10 years were 0.85, 0.85, and 0.84 and 0.79, 0.77, and 0.74, respectively, as compared with 0.81, 0.79, and 0.76 for Kaplan-Meier prognostication using the 8th edition of the TNM staging system.
We have developed parsimonious models for predicting the absolute risks of metastatic death from choroidal melanoma that take into account competing causes of death and which compare favorably with the current version of the TNM staging system. There is a need for further studies to validate the use of these models in situations where use of the TNM or LUMPO is not possible.
Proton beam radiotherapy and plaque brachytherapy are commonly applied in primary uveal melanoma (UM); however, their effect on chromosome 3 classification of UM by microsatellite analysis (MSA) for ...prognostication purposes is unknown, where the tumour is sampled post-irradiation. This study examined the prognostic accuracy of genotyping UM biopsied before or after administration of radiotherapy, by MSA.
407 UM patients treated at the Liverpool Ocular Oncology Centre between January 2011 to December 2017, were genotyped for chromosome 3 by MSA; 172 and 176 primary UM were sampled prior to and post irradiation, respectively.
Genotyping by MSA was successful in 396/407 (97%) of UM samples (196 males, 211 females; median age of 61 years (range 12 to 93) at primary treatment). There was no demonstrable association between a failure of MSA to produce a chromosome 3 classification and whether radiation was performed pre-biopsy or post-biopsy with an OR of 0.96 (95% CI 0.30 to 3.00, p=0.94). There was no evidence of association (measured as HRs) between risk of metastatic death and sampling of a primary UM before administration of radiotherapy (HR 1.1 (0.49 to 2.50), p=0.81). Monosomy 3 (HR 12.0 (4.1 to 35.0), p<0.001) was significantly associated with increased risk of metastatic death.
This study revealed that successful genotyping of UM using MSA is possible, irrespective of irradiation status. Moreover, we found no evidence that biopsy prior to radiotherapy increases metastatic mortality.
Uveal melanoma is a highly aggressive cancer of the eye, in which nearly 50% of the patients die from metastasis. It is the most common type of primary eye cancer in adults. Chromosome and mutation ...status have been shown to correlate with the disease-free survival. Loss of chromosome 3 and inactivating mutations in BAP1, which is located on chromosome 3, are strongly associated with ‘high-risk' tumors that metastasize early. Other genes often involved in uveal melanoma are SF3B1 and EIF1AX, which are found to be mutated in intermediate- and low-risk tumors, respectively. To obtain genetic information of all genes in one test, we developed a targeted sequencing method that can detect mutations in uveal melanoma genes and chromosomal anomalies in chromosome 1, 3, and 8. With as little as 10 ng DNA, we obtained enough coverage on all genes to detect mutations, such as substitutions, deletions, and insertions. These results were validated with Sanger sequencing in 28 samples. In >90% of the cases, the BAP1 mutation status corresponded to the BAP1 immunohistochemistry. The results obtained in the Ion Torrent single-nucleotide polymorphism assay were confirmed with several other techniques, such as fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, and Illumina SNP array. By validating our assay in 27 formalin-fixed paraffin-embedded and 43 fresh uveal melanomas, we show that mutations and chromosome status can reliably be obtained using targeted next-generation sequencing. Implementing this technique as a diagnostic pathology application for uveal melanoma will allow prediction of the patients' metastatic risk and potentially assess eligibility for new therapies.
The study's aim was to compare chromosome 3 aberrations of choroidal melanoma (CM) as determined by multiplex ligation dependent probe amplification (MLPA) or microsatellite analysis (MSA) in ...intraocular tumour biopsies with those results obtained from subsequent endoresection/enucleation of the same CM.
A retrospective cohort of 28 patients with CM seen between 2007 and 2014 at the Liverpool Ocular Oncology Centre was analysed. Prognostic genetic testing, for chromosome 3 status, was performed on all tumour specimens, either by MLPA or MSA, depending on DNA yield. In nine cases genetic testing was performed on a sample taken after radiotherapy; four of these had genetic information pre- and post-radiotherapy.
Fourteen biopsy specimens were analysed by MLPA and 14 by MSA. Twenty-seven endoresection or enucleation specimens were analysed by MLPA, and a single enucleation specimen by MSA. Chromosome 3 data showed prognostic concordance for the patient-matched samples in all 28 cases including 4 cases where samples were taken pre pre- and post radiotherapy. Thirteen cases were classified as monosomy 3 and 12 as disomy 3. Two cases had a loss of chromosome arm 3q in both samples and a single case showed loss of 3p in the biopsy sample with complete monosomy 3 in the subsequent enucleation sample taken 5 months later.
Intraocular biopsy of CM yields similar prognostic information to larger surgical specimens. Initial evidence, that genetic testing can be successfully conducted post radiotherapy, is also provided.
NITRO trial, ISRCTN35236442.
Unpacking the genetic etiology of uveal melanoma Thornton, Sophie; Kalirai, Helen; Aughton, Karen ...
Expert review of ophthalmology,
07/03/2020, 2020-07-03, Letnik:
15, Številka:
4
Journal Article
Recenzirano
Uveal melanoma (UM) is the most common primary intraocular tumor affecting adults. ~50% of patients will develop metastatic disease, mainly in the liver, for which there is currently no standard of ...care. The multi-faceted genetic etiology of UM informs prognosis and advises the clinical management of patients.
This review highlights the multifarious nature of the genetics that differentiate uveal nevi from UM, that initiate tumorigenesis and ultimately drive metastasis, and how these can be incorporated to multivariate models to predict whether and approximately when the metastatic disease will occur.
Despite not being able to utilize current genomic biomarkers as therapeutically actionable targets in UM at present, advances in our understanding of this cancer, may lead to their inclusion to predict response to emerging therapies. Prognostication for individual patients is likely to progress from binary classifications of high- and low-metastatic risk, utilizing a multi-factorial approach to design molecular assays. Improvement in our comprehension of the genetic etiology of UM will bring us closer to the development of effective treatments.
Uveal melanoma (UM) is the most common primary intraocular malignancy occurring in adults, with a propensity for liver metastases, which occur in ~50% of patients. Despite successful treatment of the ...primary tumour, there is currently no effective systemic therapy to treat metastatic disease. UM patient management is dependent upon accurate stratification into high or low risk of metastatic progression. Prognostication of UM patients currently involves the integration of clinical, histological, and genetic features of the tumour obtained by multiplex ligation-dependant probe amplification (MLPA) or microsatellite analysis (MSA), in order to obtain a refined predicted metastatic risk. The aims of this thesis were to examine prognostication performed in Liverpool and improve the way patients are stratified according to metastatic risk. In chapter 2 the genetic characteristics of the rarest form of UM, iris melanoma (IM) was examined. Molecular analysis was performed on archival IM samples collected over a twenty year period and demonstrated that a low-metastatic genotype was most commonly observed; this correlates with the improved survival of this UM subtype in comparison to ciliary body and choroidal melanomas. Chromosomal aberrations often seen in posterior UM are also seen in IM, in both treatment-naïve and irradiated tumours. MSA is a common method of genotyping for small UM with low DNA yields, i.e. fine-needle aspirate biopsy samples (FNAB). In chapter 3, I demonstrated that MSA can accurately determine chromosome 3 copy number in FNAB samples. I found no demonstrable differences in the quality or clarity of the genotypes generated in both treatment-naïve and irradiated UM. By examining metastasis-free survival in these samples, I established that there was no evidence that surgical intervention causes iatrogenic dissemination. Loss of 3q was identified as an indicator of poor prognosis and allelic imbalance as an indicator of good prognosis. In chapter 4, in the largest study to date, I demonstrated that loss of nuclear BAP1 protein expression (nBAP1) was a significant independent prognostic parameter associated with metastatic risk and reduced survival, with a stronger association with poor outcome than monosomy 3 (M3). Additionally I identified two subgroups within M3 UM whereby M3 nBAP1+ cases were associated with a prolonged survival in comparison to nBAP1- UM. To address the clinical need to integrate copy number analysis of chromosomes 1, 3, 6 and 8 and gene mutation status of GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CYSTLR2, PLCB4, TTC28, KTN1, TP53BP1, and CSMD1 in chapter 5, I designed, compared and validated targeted next-generation sequencing (NGS) panels for UM. I demonstrated the superiority of hybrid capture as an enrichment method, and was able to successfully analyse copy number and single nucleotide variants in both fresh and formalin fixed tissue. Mutations in BAP1 were found to be the most important prognostic factor in terms of survival, and gains of 1q were associated with an increased incidence of metastatic death. I identified novel mutations in PLCB4, TTC28, CSMD1, KTN1 and TP53BP1 that require further evaluation. In chapter 6 I investigated protein expression levels of a recently identified molecule RasGRP3 in GNAQ/GNA11 mutant UM cell lines and primary tumours and compared this with BRAF mutant conjunctival melanoma specimens. I demonstrated that RasGRP3 was significantly and selectively overexpressed in response to GNAQ/11 mutations. This demonstrates that activation of the MAP-kinase pathway occurs through a different mechanism to that occurring in conjunctival melanoma. In conclusion, I have examined in detail ways of enhancing our current practice for prognostication in UM. There is a need for BAP1 mutations and nBAP1 negativity to be implemented into routine prognostication. This may be achievable by replacing current MLPA and MSA testing with the targeted UM NGS panel.
Iris melanomas (IM) are rare and have a lower mortality than posterior uveal melanomas (UM). Our aims were to determine the prevalence of genetic changes associated with prognosis of posterior UM, in ...both treated and non-treated IM.
Retrospective database review and molecular analysis of all patients diagnosed with IM at the Liverpool Ocular Oncology Centre (LOOC) between 1993 and 2015. Archival pathology specimens of confirmed IM cases were analysed for chromosomal alterations, using multiplex ligation-dependent probe amplification (MLPA) or microsatellite analysis (MSA) depending on DNA yield, and
mutation status.
5189 patients were diagnosed with intraocular melanoma at LOOC from 1993 to 2015. Of these, 303 (5.8%) patients were diagnosed with IM. Tissue samples were available for 26 IM cases. Twelve of these cases had biopsies taken post-proton beam radiotherapy (PBR). Histological subtyping showed 14 IM being spindle, 2 epithelioid and 10 were of mixed cell type. Twenty of the 26 IM cases (77%) analysed genetically were classified as either disomy 3 (n=16) or monosomy 3 (n=4). Chromosome 6p gain was detected in 4/18 (22%) IM, and polysomy 8q in 6%.
mutations were not detected in any of the four IM cases examined. One patient with IM died from metastatic disease: this tumour was disomy 3 with 6p and 8q gains. All other patients were alive with no evidence of metastases at study closure.
Chromosomal aberrations seen in posterior UM can also be demonstrated using MLPA or MSA in both treatment naïve and PBR-treated IM. Most IM display a low-metastatic risk chromosomal profile.
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