Margherita Nannini

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.

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.

A distinct pediatric-type gastrointestinal stromal tumor in adults: potential role of succinate dehydrogenase subunit A mutations.

We read with considerable interest the article by Rege et al published in the April 2011 issue describing the histologic and clinical features of a small group of patients with gastrointestinal stromal tumors (GISTs) suggesting a new disease entity called “pediatric type.” In particular, they reported data on GISTs primarily arising from the stomach with a mean size of 5.4 cm and mainly affecting women with a median age of 31.5 years. Microscopically, they showed a predominant mixed epithelioid and spindle cell morphology with a multinodular architecture. On the basis of immunohistochemistry all tumors were positive for KIT, and 82% of cases were also positive for DOG1. Molecular analysis disclosed that they were wild Conflicts of Interest and Source of Funding: This research was supported in part by NCI grant R01CA120316. The authors have disclosed that they have no significant relationship with, or financial interest in, any commercial companies pertaining to this article. type (WT) for KIT and Alpha-type platelet-derived growth factor receptor (PDGFRA) mutations (KIT/PDGFRA WT). Clinically, the tumors often give rise to lymph node metastases, both at the time of diagnosis or as sites of distant recurrence. None of the patients showed a radiologic response to imatinib; 2 patients showed a response to sunitinib, and most of them experienced an indolent clinical course. As is well known, about 10% to 15% of adult GISTs are KIT/ PDGFRA WT and differ from mutant GISTs in clinical behavior and underlying genomic background, representing a distinct molecular subtype of GIST. However, the clinical outcome is heterogenous also among adult KIT/PDGFRA WT GIST patients, suggesting a small group with disease different from conventional adult KIT/PDGFRA WT GIST. Rege et al drew attention to “adult” GIST patients with the same histologic and clinical features as “pediatric” GIST patients sharing the absence of KIT and PDGFRA gene mutations. To date, the oncogenic defects responsible for the development of this type of disease have not been identified. We are contributing to the interesting topic highlighted by Rege et al in 2 ways: reporting our experience with 4 nonsyndromic adult GIST patients with pediatric characteristics and discussing the opportunity to explore the genomic background further, focusing particularly on succinate dehydrogenase complex (subunits A, B, C, and D) mutations. We report the data from 4 patients with a GIST localized in the stomach, which was multifocal in 2 patients, with a female predominance (3 patients out 4) and an indolent course even if metastatic (3 patients). The sites of metastases were liver and lymph nodes in 2 patients and liver, lymph nodes, and lung in 1 patient, with histologic confirmation of all sites of metastases (Table 1). Three primary tumors had a mixed morphology of spindle and epithelioid cells, and 1 had epithelioid cells with dimension ranging between 6 and 10.5 cm and a mitotic rate ranging from 1 to 10/50 high-power fields. Three tumors were classified as having high risk of recurrence according to the Miettinen Classification. The largest tumor from patient 4 had a low risk of metastatic recurrence according to the Miettinen Classification and intermediate risk according to the Fletcher Classification. However, after 13 years, the patient presented with novel lesions (dimension between 1.6 and 4.5 cm) in the stomach. Some of them were probably new primary tumors, confirmed as GIST at biopsies because they arose far from the site of the primary tumor resection, and 1 lesion was probably local recurrence because it arose at the same previous site even if it could not be confirmed or excluded. All 4 tumors were positive for KIT, whereas 3 of 4 showed immunoreactivity for CD34. DOG1 was available in only 2 cases and was positive. Two tumors were weakly positive for smooth muscle actin. None was positive for S-100 protein or desmin. None of the GISTs harbored any KIT or PDGFRA gene mutations. Two patients (1 and 3) received imatinib for metastatic disease and then experienced a prolonged disease stabilization under sunitinib treatment. Patient 2 was operated upon for primary GIST, liver, and lymph node metastases up front and received imatinib in the adjuvant setting. The last patient (patient 4) did not receive imatinib treatment; she only underwent resection of primary GIST (classified as low risk of metastatic recurrence). No data are available on the follow-up treatment of the recent new lesions. All 4 of our patients harbor mutations in succinate dehydrogenase complex, subunit A (SDHA) (Fig. 1). SDHA mutations in 2 of them were identified using a massively parallel sequencing approach and were reported in a previous article. Both of these cases have already been reported, and the 2 new ones described here were completely resequenced by conventional Sanger sequencing using specific primer pairs designed to amplify each of the 15 exons of SDHA gene without amplifying the pseudogenes located on chromosomes 3 and 5. Patient 1 was homozygous for a Letters to the Editor Am J Surg Pathol Volume 35, Number 11, November 2011

Experimental results and related clinical implications of PET detection of epidermal growth factor receptor (EGFr) in cancer

The epidermal growth factor receptor (EGFr) is one of the most studied molecules as a target for cancer therapy. Over these last few years, several studies attempting to identify predictive biomarkers of treatment response, such as the receptor status or other molecules related to the downstream signalling pathway, have been conducted. However, from a clinical point of view, the information obtained from ex vivo analyses still has various limitations that may be overcome by the combination with molecular imaging technologies which may provide a noninvasive, global, in vivo evaluation of the molecular tumour background. The aim of this review is to report the preclinical results of all positron emission tomography (PET) tracers synthesized until now for in vivo detection of EGFr in cancer. Two classes of PET compounds have been developed: labelled small molecules such as tyrosine kinase inhibitors and labelled monoclonal antibodies. The in vitro and in vivo results of these PET tracers are very different depending on the chemical properties, positron emission radionuclide, or animal models. As a consequence, various critical questions are still open, and the implications of a translation in the clinical setting for EGFr imaging in cancer patients is discussed.

Quadruple wild‐type (WT) GIST: defining the subset of GIST that lacks abnormalities of KIT, PDGFRA, SDH, or RAS signaling pathways

A subset of GISTs lack mutations in the KIT/PDGFRA or RAS pathways and yet retain an intact succinate dehydrogensase (SDH) complex. We propose that these KIT/PDGFRA/SDH/RAS‐P WT GIST tumors be designated as quadruple wild‐type (WT) GIST. Further molecular and clinicophatological characterization of quadruple WT GIST will help to determine their prognosis as well as assist in the optimization of medical management, including clinical test of novel therapies.

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.Mutation of tyrosine kinase receptors is a mechanism of drug resistance that can occur either at the beginning of treatment (primary resistance) or during the course of therapy (secondary resistance). In addition, mutational status can predict the response to treatment with tyrosine kinase inhibitors, but the role of mutational status as a prognostic factor remains controversial.Evidence of a potential role of mutational status as a prognostic factor has emerged over the past decade. The presence of KIT exon 11 insertion/deletion involving either one or both Trp557-Lys558 amino acids correlates with a poorer clinical outcome if compared to patients with tumors wild type for KIT exon 11 mutations. A malignant clinical behavior has also been documented for KIT exon 13 and KIT exon 9 mutant GIST. Patients with GIST harboring a PDGFRA mutation seem to have a better prognosis than the others.The aim of this paper is to review the clinical significance of tyrosine kinase mutational status.