Pitfalls in mutational testing and reporting of common KIT and PDGFRA mutations in gastrointestinal stromal tumors

<p>Abstract</p> <p>Background</p> <p>Mutation analysis of <it>KIT </it>and <it>PDGFRA </it>genes in gastrointestinal stromal tumors is gaining increasing importance for prognosis of GISTs and for prediction of treatment response. Several groups have identified specific mutational subtypes in <it>KIT </it>exon 11 associated with an increased risk of metastatic disease whereas GISTs with <it>PDGFRA </it>mutations often behave less aggressive. Furthermore, in advanced GIST disease with proven <it>KIT </it>exon 9 mutation the doubled daily dose of 800 mg imatinib increases the progression free survival and is now recommended both in the European and the American Guidelines. In Germany, there are still no general rules how to perform mutational analysis.</p> <p>Methods</p> <p>When comparing results from six different molecular laboratories we recognized the need of standardisation. Six German university laboratories with experience in mutation analysis in GISTs joined together to develop recommendations for the mutation analysis of the most common and clinically relevant hot spots, i. e. <it>KIT </it>exons 9 and 11 and <it>PDGFRA </it>exon 18. We performed a three-phased interlaboratory trial to identify pitfalls in performing molecular analysis in GISTs.</p> <p>Results</p> <p>We developed a design for a continuous external laboratory trial. In 2009 this external trial was conducted by 19 laboratories via the initiative for quality assurance in pathology (QuiP) of the German Society of Pathology and the Professional Association of German Pathologists.</p> <p>Conclusions</p> <p>By performing a three-phased internal interlaboratory trial and conducting an external trial in Germany we were able to identify potential pitfalls when performing KIT and PDGFRA mutational analysis in gastrointestinal stromal tumors. We developed standard operation procedures which are provided with the manuscript to allow other laboratories to prevent these pitfalls.</p

Perioperative Management of Extremity Soft Tissue Sarcomas

Biological, physical and clinical aspects of cancer treatment with ionising radiatio

P53 expression is significantly correlated with high risk of malignancy and epithelioid differentiation in GISTs. An immunohistochemical study of 104 cases

<p>Abstract</p> <p>Background</p> <p>Molecular analyses of the <it>c-kit </it>and <it>PDGFRα </it>genes have contributed greatly to our understanding of the development of gastrointestinal stromal tumors (GISTs), but little is known about their malignant potential. The aim of our study was to evaluate cell cycle regulators as potential prognostic markers in GISTs.</p> <p>Methods</p> <p>We investigated 104 KIT positive GISTs from various tumor sites in immunoassays on CD34, Ki67 and particularly on P53, BCL-2 and Cyclin D1. The results were compared with tumor size, mitotic rate, proliferative activity, histological subtype, nuclear atypia and risk assessment according to Fletcher and Miettinen. Occurrence of metastases and survival were also taken into account.</p> <p>Results</p> <p>The expression of P53 was significantly correlated with high risk criteria towards malignancy and epithelioid differentiation in GISTs. Likewise P53 label correlated significantly with the established prognostic indicators: tumor size, mitotic rate, nuclear atypia and proliferative activity. Regarding the site of tumor presentation, P53 was not a decisive factor. BCL-2 and Cyclin D1 expression was not related to any of the prognostic indicators.</p> <p>Conclusion</p> <p>The present data identified P53 being a recommendable marker for predicting the risk of malignancy in GISTs. In addition, we found P53 significantly correlated with epithelioid tumor differentiation, independent of tumor site. BCL-2 and Cyclin D1, however, did not prove to be deciding markers for diagnosis and prognosis.</p

Strong evidence that the common variant S384F in BRCA2 has no pathogenic relevance in hereditary breast cancer

INTRODUCTION: Unclassified variants (UVs) of unknown clinical significance are frequently detected in the BRCA2 gene. In this study, we have investigated the potential pathogenic relevance of the recurrent UV S384F (BRCA2, exon 10). METHODS: For co-segregation, four women from a large kindred (BN326) suffering from breast cancer were analysed. Moreover, paraffin-embedded tumours from two patients were analysed for loss of heterozygosity. Co-occurrence of the variant with a deleterious mutation was further determined in a large data set of 43,029 index cases. Nature and position of the UV and conservation among species were evaluated. RESULTS: We identified the unclassified variant S384F in three of the four breast cancer patients (the three were diagnosed at 41, 43 and 57 years of age). One woman with bilateral breast cancer (diagnosed at ages 32 and 50) did not carry the variant. Both tumours were heterozygous for the S384F variant, so loss of the wild-type allele could be excluded. Ser384 is not located in a region of functional importance and cross-species sequence comparison revealed incomplete conservation in the human, dog, rodent and chicken BRCA2 homologues. Overall, the variant was detected in 116 patients, five of which co-occurred with different deleterious mutations. The combined likelihood ratio of co-occurrence, co-segregation and loss of heterozygosity revealed a value of 1.4 × 10(-8 )in favour of neutrality of the variant. CONCLUSION: Our data provide conclusive evidence that the S384F variant is not a disease causing mutation

Integrative DNA methylation and gene expression analysis in high-grade soft tissue sarcomas

BACKGROUND: High-grade soft tissue sarcomas are a heterogeneous, complex group of aggressive malignant tumors showing mesenchymal differentiation. Recently, soft tissue sarcomas have increasingly been classified on the basis of underlying genetic alterations; however, the role of aberrant DNA methylation in these tumors is not well understood and, consequently, the usefulness of methylation-based classification is unclear. RESULTS: We used the Infinium HumanMethylation27 platform to profile DNA methylation in 80 primary, untreated high-grade soft tissue sarcomas, representing eight relevant subtypes, two non-neoplastic fat samples and 14 representative sarcoma cell lines. The primary samples were partitioned into seven stable clusters. A classification algorithm identified 216 CpG sites, mapping to 246 genes, showing different degrees of DNA methylation between these seven groups. The differences between the clusters were best represented by a set of eight CpG sites located in the genes SPEG, NNAT, FBLN2, PYROXD2, ZNF217, COL14A1, DMRT2 and CDKN2A. By integrating DNA methylation and mRNA expression data, we identified 27 genes showing negative and three genes showing positive correlation. Compared with non-neoplastic fat, NNAT showed DNA hypomethylation and inverse gene expression in myxoid liposarcomas, and DNA hypermethylation and inverse gene expression in dedifferentiated and pleomorphic liposarcomas. Recovery of NNAT in a hypermethylated myxoid liposarcoma cell line decreased cell migration and viability. CONCLUSIONS: Our analysis represents the first comprehensive integration of DNA methylation and transcriptional data in primary high-grade soft tissue sarcomas. We propose novel biomarkers and genes relevant for pathogenesis, including NNAT as a potential tumor suppressor in myxoid liposarcomas

Chromosome copy number changes carry prognostic information independent of KIT/PDGFRA point mutations in gastrointestinal stromal tumors

<p>Abstract</p> <p>Background</p> <p>Oncogenic point mutations in <it>KIT </it>or <it>PDGFRA </it>are recognized as the primary events responsible for the pathogenesis of most gastrointestinal stromal tumors (GIST), but additional genomic alterations are frequent and presumably required for tumor progression. The relative contribution of such alterations for the biology and clinical behavior of GIST, however, remains elusive.</p> <p>Methods</p> <p>In the present study, somatic mutations in <it>KIT </it>and <it>PDGFRA </it>were evaluated by direct sequencing analysis in a consecutive series of 80 GIST patients. For a subset of 29 tumors, comparative genomic hybridization was additionally used to screen for chromosome copy number aberrations. Genotype and genomic findings were cross-tabulated and compared with available clinical and follow-up data.</p> <p>Results</p> <p>We report an overall mutation frequency of 87.5%, with 76.25% of the tumors showing alterations in <it>KIT </it>and 11.25% in <it>PDGFRA</it>. Secondary <it>KIT </it>mutations were additionally found in two of four samples obtained after imatinib treatment. Chromosomal imbalances were detected in 25 out of 29 tumors (86%), namely losses at 14q (88% of abnormal cases), 22q (44%), 1p (44%), and 15q (36%), and gains at 1q (16%) and 12q (20%). In addition to clinico-pathological high-risk groups, patients with <it>KIT </it>mutations, genomic complexity, genomic gains and deletions at either 1p or 22q showed a significantly shorter disease-free survival. Furthermore, genomic complexity was the best predictor of disease progression in multivariate analysis.</p> <p>Conclusions</p> <p>In addition to <it>KIT/PDGFRA </it>mutational status, our findings indicate that secondary chromosomal changes contribute significantly to tumor development and progression of GIST and that genomic complexity carries independent prognostic value that complements clinico-pathological and genotype information.</p

Vatalanib for metastatic gastrointestinal stromal tumour (GIST) resistant to imatinib: final results of a phase II study

BACKGROUND: Vatalanib (PTK787/ZK 222584) inhibits a few tyrosine kinases including KIT, platelet-derived growth factor receptors (PDGFRs) and vascular endothelial growth factor receptors (VEGFRs). We report efficacy and safety results of vatalanib in advanced gastrointestinal stromal tumour (GIST) resistant to imatinib or both imatinib and sunitinib. PATIENTS AND METHODS: Forty-five patients whose metastatic GIST had progressed on imatinib were enrolled. Nineteen (42.2%) patients had received also prior sunitinib. Vatalanib 1250 mg was administered orally daily. RESULTS: Eighteen patients (40.0%; 95% confidence interval (CI), 25.7-54.3%) had clinical benefit including 2 (4.4%) confirmed partial remissions (PR; duration, 9.6 and 39.4 months) and 16 (35.6%) stabilised diseases (SDs; median duration, 12.5 months; range, 6.0-35.6+ months). Twelve (46.2%) out of the 26 patients who had received prior imatinib only achieved either PR or SD compared with 6 (31.6%, all SDs) out of the 19 patients who had received prior imatinib and sunitinib (P = 0.324). The median time to progression was 5.8 months (95% CI, 2.9-9.5 months) in the subset without prior sunitinib and 3.2 (95% CI, 2.1-6.0) months among those with prior imatinib and sunitinib (P = 0.992). Vatalanib was generally well tolerated. CONCLUSION: Vatalanib is active despite its narrow kinome interaction spectrum in patients diagnosed with imatinib-resistant GIST or with imatinib and sunitinib-resistant GIST

SLUG transcription factor : a pro-survival and prognostic factor in gastrointestinal stromal tumour

Background: The SLUG transcription factor has been linked with the KIT signalling pathway that is important for gastrointestinal stromal tumour (GIST) tumourigenesis. Its clinical significance in GIST is unknown. Methods: Influence of SLUG expression on cell proliferation and viability were investigated in GIST48 and GIST882 cell lines. The association between tumour SLUG expression in immunohistochemistry and recurrence-free survival (RFS) was studied in two clinical GIST series, one with 187 patients treated with surgery alone, and another one with 313 patients treated with surgery and adjuvant imatinib. Results: SLUG downregulation inhibited cell proliferation, induced cell death in both cell lines, and sensitised GIST882 cells to lower imatinib concentrations. SLUG was expressed in 125 (25.0%) of the 500 clinical GISTs evaluated, and expression was associated with several factors linked with unfavourable prognosis. SLUG expression was associated with unfavourable RFS both when patients were treated with surgery alone (HR = 3.40, 95% CI = 1.67-6.89, P = 0.001) and when treated with surgery plus adjuvant imatinib (HR = 1.83, 95% CI = 1.29-2.60, P = 0.001). Conclusions: GIST patients with high tumour SLUG expression have unfavourable RFS. SLUG may mediate pro-survival signalling in GISTs.Peer reviewe

Final analysis of the randomized trial on imatinib as an adjuvant in localized gastrointestinal stromal tumors (GIST) from the EORTC Soft Tissue and Bone Sarcoma Group (STBSG), the Australasian Gastro-Intestinal Trials Group (AGITG), UNICANCER, French Sarcoma Group (FSG), Italian Sarcoma Group (AGITG), UNICANCER, French Sarcoma Group (FSG), Italian Sarcoma Group (ISG), and Spanish Group for Research on Sarcomas (GEIS)

Background: In 2004, we started an intergroup randomized trial of adjuvant imatinib versus no further therapy after R0-R1 surgery in localized, high/intermediate-risk gastrointestinal stromal tumors (GIST) patients. Interim analysis results were published in 2015 upon recommendation from an independent data review committee. We report the final outcome of the study.Patients and methods: This was a randomized, open-label, multicenter phase III trial carried out at 112 hospitals in 12 countries. Patients were randomized to 2 years of imatinib, 400 mg daily, or no further therapy after surgery. The primary endpoint was imatinib failure-free survival (IFFS), while relapse-free survival (RFS), relapse-free interval (RFI), overall survival (OS) and toxicity were secondary endpoints. Adjusting for the interim analyses, results on IFFS were assessed on a 4.3% significance level; for the other endpoints, 5% was used.Results: Nine hundred and eight patients were randomized between January 2005 and October 2008: 454 to imatinib and 454 to observation; 835 patients were eligible. With a median follow-up of 9.1 years, 5 (10)-year IFFS was 87% (75%) in the imatinib arm versus 83% (74%) in the control arm [hazard ratio (HR) 0.87, 95.7% confidence interval (CI) (0.65; 1.15), P = 0.31]; RFS was 70% versus 63% at 5 years and 63% versus 61% at 10 years, [HR = 0.71, 95% CI (0.57; 0.89), P = 0.002]; OS was 93% versus 92% at 5 years and 80% versus 78% at 10 years [HR 0.88, 95% CI (0.65; 1.21), P = 0.43]. Among 526 patients with high-risk GIST by local pathology, 10-year IFFS and RFS were 69% versus 61%, and 48% versus 43%, respectively.Conclusions: With 9.1 years of follow-up, a trend toward better long-term IFFS in imatinib-treated patients was observed in the high-risk subgroup. Although the difference was not statistically significant and the surrogacy value of such an endpoint is not validated, this may be seen as supporting the results reported by the Scandinavian/German trial, showing a sustained small but significant long-term OS benefit in high-risk GIST patients treated with 3 years of adjuvant imatinib.Experimentele farmacotherapi

The role of mutational analysis of KIT and PDGFRA in gastrointestinal stromal tumors in a clinical setting

Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract. Most GIST harbor a mutation affecting either the KIT or PDGFRA genes, whereas a small subgroup of tumors is wild type for mutations