Maria Abbondanza Pantaleo

MiR-101 downregulation is involved in cyclooxygenase-2 overexpression in human colon cancer cells.

Overexpressed cyclooxygenase-2 (COX-2) strongly contributes to the growth and invasiveness of tumoral cells in patients affected by colorectal cancer (CRC). It has been demonstrated that COX-2 overexpression depends on different cellular pathways involving both transcriptional and post-transcriptional regulations. We assumed that COX-2 expression could be regulated also by microRNAs (miRNAs) since these short RNA molecules participate to the fine regulation of several genes implicated in cell growth and differentiation. In this paper, we report the inverse correlation between COX-2 and miR-101 expression in colon cancer cell lines and we demonstrated in vitro the direct inhibition of COX-2 mRNA translation mediated by miR-101. Moreover, this correlation was supported by data collected ex vivo, in which colon cancer tissues and liver metastases derived from CRC patients were analyzed. These findings provide a novel molecular insight in the modulation of COX-2 at post-transcriptional level by miR-101 and strengthen the observation that miRNAs are highly implicated in the control of gene expression. An impairment of miR-101 levels could represent one of the leading causes of COX-2 overexpression in colon cancer cells.

SDHA Loss-of-Function Mutations in KIT – PDGFRA Wild-Type Gastrointestinal Stromal Tumors Identified by Massively Parallel Sequencing

Approximately 10%-15% of gastrointestinal stromal tumors (GISTs) in adults do not harbor any mutation in the KIT or PDGFRA genes (ie, KIT/PDGFRA wild-type GISTs). Recently, mutations in SDHB and SDHC (which encode succinate dehydrogenase subunits B and C, respectively) but not in SDHA and SDHD (which encode subunits A and D, respectively) were identified in KIT/PDGFRA wild-type GISTs. To search for novel pathogenic mutations, we sequenced the tumor transcriptome of two young adult patients who developed sporadic KIT/PDGFRA wild-type GISTs by using a massively parallel sequencing approach. The only variants identified as disease related by computational analysis were in SDHA. One patient carried the homozygous nonsense mutation p.Ser384X, the other patient was a compound heterozygote harboring a p.Arg31X nonsense mutation and a p.Arg589Trp missense mutation. The heterozygous nonsense mutations in both patients were present in germline DNA isolated from peripheral blood. Protein structure analysis indicates that all three mutations lead to functional inactivation of the protein. This is the first report, to our knowle dge, that identifies SDHA inactivation as a common oncogenic event in GISTs that lack a mutation in KIT and PDGFRA.

Hypersensitivity reactions related to oxaliplatin (OHP)

Patients treated with platinum compounds are subject to hypersensitivity reactions. Our study has highlighted the reactions related to oxaliplatin (OHP) infusion. One hundred and twenty-four patients affected by advanced colorectal cancer were treated with different schedules containing OHP, at the Institute of Haematology and Medical Oncology ‘L. and A. Seragnoli’ of Bologna and at the Medical Oncology Division of Livorno Hospital. Seventeen patients (13%) showed hypersensitivity reactions after a few minutes from the start of the OHP infusion. Usually, these reactions were seen after 2–17 exposures to OHP (Mean±s.e.: 9.4±1.07). No patient experienced allergic reactions at his/her first OHP infusion. Eight patients developed a mild reaction consisting of flushing and swelling of the face and hands, itching, sweating and lachrymation. The remaining nine patients showed a moderate–severe reaction with dyspnoea, wheezing, laryngospasm, psycho-motor agitation, tachycardia, precordial pain, diffuse erythema, itching and sweating. Six patients out of 17 were re-exposed to the drug with premedication of steroids and all except one developed the hypersensitivity reaction again. The cumulative dose, the time of exposure to OHP and the clinical features are variable and unpredictable. The risk of developing hypersensitivity reactions in patients treated with a short infusion of OHP cannot be underestimated.

Gene expression profiling in colorectal cancer using microarray technologies: results and perspectives.

Nowadays molecular biology represents one of the most interesting topics in medical oncology, because it provides a global and detailed view on the molecular changes involved in tumour progression, leading to a better understanding of the carcinogenesis process, to discovering new prognostic markers and novel therapeutic targets. The gene expression profiling analysis with microarray technology has shown a great potential in cancer research and in medical oncology, mapping simultaneously the expression of thousands of genes in a single tumour sample and giving a measurement of articulated genes expression patterns. Colorectal cancer represents a wide and exciting area of research for molecular biology, due to the growing need of a molecular classification as well as prognostic and predictive molecular factors that may guide oncologists in patients clinical management. The aim of this review is to analyze the state of art of gene expression profile in colorectal cancer using microarrays technologies and to explore some perspectives in this research field.

A molecular portrait of gastrointestinal stromal tumors: an integrative analysis of gene expression profiling and high-resolution genomic copy number

In addition to KIT and PDGFRA mutations, sequential accumulation of other genetic events is involved in the development and progression of gastrointestinal stromal tumors (GISTs). Until recently, the significance of these other alterations has not been thoroughly investigated. We report the first study that integrates gene expression profiling and high-resolution genomic copy number analyses in GIST. Fresh tissue specimens from 25 patients with GIST were collected, and gene expression profiling and high-resolution genomic copy number analyses were performed, using Affymetrix U133Plus and SNP array 6.0. We found that all 21 mutant GIST patients showed both macroscopic cytogenetic alterations and cryptic microdeletions or amplifications, whereas 75% (three of four) of wild-type patients with GIST did not show genomic imbalances. The most frequently observed chromosomal alterations in patients with mutant GIST included 14q complete or partial deletion (17 of 25), 1p deletion (14 of 25) and 22q deletion (10 of 25). Genetic targets of the chromosomal aberrations were selected by integrated analysis of copy number and gene expression data. We detected the involvement of known oncogenes and tumor suppressors including KRAS in chr 12p amplification and KIF1B, PPM1A, NF2 in chr 1p, 14q and 22p deletions, respectively. The genomic segment most frequently altered in mutated samples was the 14q23.1 region, which contains potentially novel tumor suppressors, including DAAM1, RTN1 and DACT1. siRNA-mediated RTN1 downregulation showed evidence for the potential role in GIST pathogenesis. The combination of gene expression profiling and high-resolution genomic copy number analysis offers a detailed molecular portrait of GISTs, providing an essential comprehensive knowledge necessary to guide the discovery of novel target genes involved in tumor development and progression.

Treatment of Brain Metastases of Malignant Melanoma with Temozolomide

To the Editor: A 57-year-old man presented with an ulcerative, acral–lentiginous lesion on the sole of the left foot. Pathological examination showed a melanoma (Breslow depth, 2.2 mm; Clark level ...

Insulin-like growth factor 1 receptor expression in wild-type GISTs: A potential novel therapeutic target

Aberrations of the Insulin‐like Growth Factor (IGF) system have been found in association with a variety of cancer types. The potential role of IGF1R has been postulated in a small subset of GISTs, but until now the implications of its aberrations have not been defined. The aim of the study was to examine the IGF1R status in patients with gastric GIST in regard to KIT and PDGFRA genotype. Fresh resection specimens were collected from 8 primary tumours [2 wild‐type (WT) and 6 mutant cases]. IGF1R was studied as gene expression profiling with Affymetrix GeneChip HG‐U133Plus 2.0 arrays and as genomic copy number with SNP array analysis Affymetrix Genome Wide Human SNP 6.0 arrays, and at protein level with western blotting (WB) and immunohistochemistry (IHC). The unsupervised analysis of gene expression profiling of our patients merged with a data set from gastric GISTs identified 2 patients out of 8 with different expression of IGF1R. The data were confirmed by WB and IHC. In particular, IGF1R was upregulated in 2 young patients (<30‐years old), who had both WT disease and metastases at diagnosis. The SNP array analysis showed that none of the tumours had IGF1R amplification. GISTs are characterized by abnormalities of the KIT and PDGFRA receptors that affect prognosis and response to tyrosine kinase inhibitors. Both young adult with WT GIST had the over‐expression of IGF1R at mRNA and protein level. These results further confirm the hypothesis that IGF1R may be a potential therapeutic target in GISTs lacking KIT and PDGFRA mutations.

Analysis of all subunits, SDHA, SDHB, SDHC, SDHD, of the succinate dehydrogenase complex in KIT/PDGFRA wild-type GIST.

Mutations of genes encoding the subunits of the succinate dehydrogenase (SDH) complex were described in KIT/PDGFRA wild-type GIST separately in different reports. In this study, we simultaneously sequenced the genome of all subunits, SDHA, SDHB, SDHC, and SDHD in a larger series of KIT/PDGFRA wild-type GIST in order to evaluate the frequency of the mutations and explore their biological role. SDHA, SDHB, SDHC, and SDHD were sequenced on the available samples obtained from 34 KIT/PDGFRA wild-type GISTs. Of these, in 10 cases, both tumor and peripheral blood (PB) were available, in 19 cases only tumor, and in 5 cases only PB. Overall, 9 of the 34 patients with KIT/PDGFRA wild-type GIST carried mutations in one of the four subunits of the SDH complex (six patients in SDHA, two in SDHB, one in SDHC). WB and immunohistochemistry analysis showed that patients with KIT/PDGFRA wild-type GIST who harbored SDHA mutations exhibited a significant downregulation of both SDHA and SDHB protein expression, with respect to the other GIST lacking SDH mutations and to KIT/PDGFRA-mutated GIST. Clinically, four out of six patients with SDHA mutations presented with metastatic disease at diagnosis with a very slow, indolent course. Patients with KIT/PDGFRA wild-type GIST may harbor germline and/or de novo mutations of SDH complex with prevalence for mutations within SDHA, which is associated with a downregulation of SDHA and SDHB protein expression. The presence of germline mutations may suggest that these patients should be followed up for the risk of development of other cancers.

An overview on molecular biology of KIT/PDGFRA wild type (WT) gastrointestinal stromal tumours (GIST)

Background About 85% of paediatric gastrointestinal stromal tumours (GISTs) and about 10–15% of adult GISTs do not harbour any mutations in the KIT and PDGFRA genes and are defined as KIT/PDGFRA wild type (WT). Over the years it has been demonstrated that KIT/PDGFRA WT GISTs are profoundly different from mutant GIST in clinical and molecular profiles, so that they are now considered a separate pathological entity. Moreover, due to their extreme molecular and clinical heterogeneity, KIT/PDGFRA WT GIST should be considered as a family of diseases and not as a single disease entity. However, although several genetic alterations belonging only to KIT/PDGFRA WT GIST have been identified, the exact role of these molecules in the pathogenesis and development of this subgroup is not yet defined. Methods The aim of this review is to report all current data about the molecular biology of syndromic and non-syndromic KIT/PDGFRA WT GIST, focusing on the potential clinical implication of each biological feature shared by this subgroup and discussing unresolved problems and future research perspectives on this topic. Results WT GIST is definitely a set of different diseases sustained by specific molecular alterations not yet completely known. Conclusion Large series of patients are required for defining the biological fingerprint of each subtype and integrating it with clinical data. This will allow the transfer of biological information to clinical practice and its use as an additional tool for diagnosis, prognosis and selection of medical treatment.

Integrated genomic study of quadruple-WT GIST ( KIT/PDGFRA/SDH/RAS pathway wild-type GIST)

BackgroundAbout 10-15% of adult gastrointestinal stromal tumors (GIST) and the vast majority of pediatric GIST do not harbour KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutations (J Clin Oncol 22:3813–3825, 2004; Hematol Oncol Clin North Am 23:15–34, 2009). The molecular biology of these GIST, originally defined as KIT/PDGFRA wild-type (WT), is complex due to the existence of different subgroups with distinct molecular hallmarks, including defects in the succinate dehydrogenase (SDH) complex and mutations of neurofibromatosis type 1 (NF1), BRAF, or KRAS genes (RAS-pathway or RAS-P).In this extremely heterogeneous landscape, the clinical profile and molecular abnormalities of the small subgroup of WT GIST suitably referred to as quadruple wild-type GIST (quadrupleWT or KITWT/PDGFRAWT/SDHWT/RAS-PWT) remains undefined. The aim of this study is to investigate the genomic profile of KITWT/PDGFRAWT/SDHWT/RAS-PWT GIST, by using a massively parallel sequencing and microarray approach, and compare it with the genomic profile of other GIST subtypes.MethodsWe performed a whole genome analysis using a massively parallel sequencing approach on a total of 16 GIST cases (2 KITWT/PDGFRAWT/SDHWT and SDHBIHC+/SDHAIHC+, 2 KITWT/PDGFRAWT/SDHAmut and SDHBIHC-/SDHAIHC- and 12 cases of KITmut or PDGFRAmut GIST). To confirm and extend the results, whole-genome gene expression analysis by microarray was performed on 9 out 16 patients analyzed by RNAseq and an additional 20 GIST patients (1 KITWT/PDGFRAWTSDHAmut GIST and 19 KITmut or PDGFRAmut GIST). The most impressive data were validated by quantitave PCR and Western Blot analysis.ResultsWe found that both cases of quadrupleWT GIST had a genomic profile profoundly different from both either KIT/PDGFRA mutated or SDHA-mutated GIST. In particular, the quadrupleWT GIST tumors are characterized by the overexpression of molecular markers (CALCRL and COL22A1) and of specific oncogenes including tyrosine and cyclin- dependent kinases (NTRK2 and CDK6) and one member of the ETS-transcription factor family (ERG).ConclusionWe report for the first time an integrated genomic picture of KITWT/PDGFRAWT/SDHWT/RAS-PWT GIST, using massively parallel sequencing and gene expression analyses, and found that quadrupleWT GIST have an expression signature that is distinct from SDH-mutant GIST as well as GIST harbouring mutations in KIT or PDGFRA. Our findings suggest that quadrupleWT GIST represent another unique group within the family of gastrointestintal stromal tumors.