Early Days Research

The university training: biology and genetics (1994-1998)

I started my training at the University of Genova (Italy), where in 1995 I was awarded a degree in Biological Sciences (Molecular Biology). The course had just been reformed and included 27 exams, 2 practical lab courses and an exam in English language; this five years course gave me a broad formation in basic science and biology in particular. Since the beginning of my academic career, I have been fascinated by genetics and, over the years, my main research interests have developed in this field.

During my early research training in genetics, I worked on fragile X syndrome [1] and I was involved in the cloning of the SH3BGR gene on chromosome 21q22.3 [2]. My specialization (PhD equivalent) research project was focused on the molecular characterization of human syndromes associated with defects in nucleotide excision repair (NER), including xeroderma pigmentosum (XP) and Cockayne Syndrome (CS) [3, 4, 6]. These early years were key in developing the analytical skills needed for scientific research and I was lucky to be exposed to many different general and molecular biology techniques (including cell culture, nucleic acids extraction, Southern and Northern blotting, PCR, miniprep, cloning, sequencing). In those years, I also become familiar with sequence analysis through online databases learning the basis of the what was then the beginning of bioinformatics. I also received formal training in bioinformatics during a week long course at the International Centre for Genetic Engineering and Biotechnology (Trieste, Italy).

Some years ago, with the Human Genome Project under way, I developed a great interest in genomics and I worked on a very early cancer genomics project at the International Agency for Cancer Research (World Health Organization, Lyon, France). As the new microarrays technologies were developing faster in the USA, I joined Prof. Krahe’s laboratory at the Ohio State University (Columbus, OH, USA) and a year later moved with him to the University of Texas M.D. Anderson Cancer Centre (Houston, TX, USA). In 2004, I moved back to Europe joining the genomics laboratory at the Wellcome Trust Centre for Human Genetics of the University of Oxford (Oxford, UK).

The early years: from genetics to genomics (1998 - 2000)

Working on DNA repair for my thesis, I developed an interest in cancer genetics. I was awarded a post-doctoral training fellowship to work at the International Agency for Research on Cancer (IARC, WHO, Lyon, France) and genomics was developing in this field (oncogenomics) at the time.

Analysis of the role of protein phosphatase 2A (A-alpha and A-beta subunits) in human gliomas [9]

In the Unit of Molecular Pathology at IARC my work on protein phosphatase 2A (PP2A, A-alpha and A-beta subunits) in human gliomas revealed a lack of mutations and the presence of alterations in the A-alpha subunit protein levels. Mutations in these subunits had been reported in other cancer types, thus I developed and implemented single strand conformational polymorphism (SSCP) analysis to screen mutations in the genes coding for the two proteins. Following the changes identified in SSCP by DNA sequencing revealed only the presence of a few polymorphisms; on the other hand studies at the protein level showed that in 43% of the tumors the level of the Aa subunit was reduced at least 10-fold. At the same time the levels of the B-alpha and C-alpha subunits were normal. The study showed that in these brain tumor samples very low levels of core and holoenzyme are associated with high amounts of unregulated catalytic C subunit. This imbalance could have a role in cell proliferation acting through the Wnt signaling pathway.

Expression profiling of low grade gliomas [7]

In 1999 I had the first chance to be directly involved in an oncogenomics research project and I joined in a project generating microarrays based gene expression profiles of diffuse astrocytomas. I was involved in particular in the analysis of the results and this was the first experience of the complexity involved in microarrays data analysis. I also contributed to the development of a serie of semi-quantitative PCR assays to validate at the mRNA level the changes observed in the microarrays experiment.

Technology transfer in collaborative projects

I was also involved in the implementation of SSCP based mutation analysis methodologies for the BUB gene family [8] and in microsatellite based analysis loss-of-heterozygosity (LOH) studies [5] in other brain tumors. The close interactions with expert pathologists were very important to broaden my knowledge and, even though I did not receive formal training, I learnt a lot about general pathology. Furthermore, working at IARC I came to appreciate the importance of both genetics and pathology in cancer diagnosis and prognosis.

Advanced training in genomics research (2000-2004)

As I had experienced the need for further advanced training in genomics data generation and analysis, in August 2000, I joined Prof. Krahe’s laboratory, then at The Ohio State University and later followed him to University of Texas M.D. Anderson Cancer Center. During my stay in the USA, I was involved in several projects using high-throughput genomics approaches to better understand cancer biology. I also helped in the preparation of grant applications, learning from my mentor new skills in this important aspect of independent research work. Furthermore, when Dr. Krahe was appointed Associate Professor at the University of Texas M.D. Anderson Cancer Center, I had the unique opportunity to be involved in the new high-throughput laboratory set up (ABI 3100 Sequencer, Affymetrix Array System, Biotage Pyrosequencer, BioRad iCycler Real-time PCR, Agilent Bioanalyser) in Houston, thus getting a real field experience in laboratory management.

Gene expression profiling of metastatic signature in Head and Neck Squamous Cell Carcinoma (HNSCC)

I used a comprehensive genomic approach to characterize Head and Neck Squamous Cell Carcinoma (HNSCC) carcinogenesis and metastasis using a sample collection of matching normal adjacent tissue, primary tumor and lymphnode metastasis. A common gene expression pattern is present in paired primary tumors and metastases (detected using Affymetrix GeneChip Gene Expression microarrays). This observation suggests that most of the metastatic potential is inherent to the primary tumor. Nonetheless the matching samples design, allowed the identification a metastasis-specific signature of 46 genes; several of these genes have been implicated as biologically relevant in other tumor systems. Overall these data are consistent with the notion that a limited number of additional clonal changes are necessary to yield the final metastatic cell(s) [19]. The results of the gene expression project were integrated with LOH profiling using SNP arrays (Affymetrix Genotyping 10K arrays) and HPV infection status detected by real-time PCR to better understand HNSCC carcinogenesis [22]. Working on this project I had the great opportunity to interact closely with our collaborator Keith A. Baggerly, Associate Professor in the Department of Biostatistics and Applied Mathematics, gaining in depth knowledge of available statistical methods, as well as of new strategies to analyze complex datasets. This included gene ontology and pathway based analysis, that revealed the association of HNSCC progression with the dysregulation of genes involved in cell adhesion and remodeling of the extra-cellular matrix, as well as cell cycle regulation, identifying several genes never before associated with HNSCC tumorigenesis as potential novel biomarkers.

Development of novel methylation detection method using Pyrosequencing technology [10]

Aberrant DNA methylation is a common event in cancer and thus it is important to have reliable methods to detect this epigenetic event. While at MD Anderson I developed a new method to detect CpG sites methylation using Pyrosequencing technology (Biotage, Sweden). PyroMethA (Pyrosequencing Methylation Analysis) is a modification of the Combined Bisulfite Restriction Analysis (COBRA), where the restriction analysis after the bisulfite treatment is substituted with the highly quantitative Pyrosequencing reaction. The new method is rapid, highly quantitative and suitable for high-throughput studies. To further improve the applicability I implemented a universal primer approach that allows substantial experimental cost reduction.

Genomics data analysis in collaborative projects.

Part of my research work included frequent interactions and collaboration with computer programmers in the group; I have contributed to the design of several Perl-based tools for the analysis of complex datasets. Working in the laboratory, I also contributed to a very interesting genome-wide LOH study in Wilms Tumor to identify novel tumor suppressor genes [11]. In addition I shared my expertise in expression profiling data analysis, contributing to different projects in collaboration with other groups [12, 15]. During these years I had also many opportunity to be involved in the training of junior post-doctoral fellows, doctoral students and technicians, an activity that I enjoyed very much.

Research and Development in genomics (2004-2007)

In 2004 I joined the Genomics Laboratory at the Wellcome Trust Centre for Human Genetics at the University of Oxford. The group managed several high-throughput technology platforms, including at that time: Affymetrix and Illumina Microarrays platforms for gene expression and high-throughput genotyping, custom microarrays (for protein DNA interaction, gene expression, microRNAs and mutation detection), Sequenom Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight mass spectrometry (MALDI-TOF MS for genotyping, gene expression and DNA methylation analysis), Applied Biosystems 3700 sequencer, the BioRad iCycler real-time PCR and the Agilent Bioanalyser for RNA QC.

DNA methylation analysis using MALDI-TOF mass spectrometry

Epigenetics role in human disease beyond cancer is very well recognized, but not completely understood. When I joined the Genomics Laboratory one of my responsibilities was to implement, in collaboration with Sequenom, a MALDI-TOF based approach for high-throughput DNA methylation profiling. The methodology we implemented offers advantages over other approaches (see more details in the review on MALDI-TOF technology [14]) and several collaborations using this technology to characterize the methylation status of genomic regions of interest in several diseases were developed and some are still in progress [17, 23].

Validation of a newly developed algorithm for the detection of copy number variants using SNP arrays

In the last few years copy number variants (CNVs) have been identified in the human genome and there is a growing interest in the detection of such variants to understand their role in gene regulation and the possible association of specific variants with human disease. In the last year I was involved in a project to validate the new BeadArray™ Infinium™ (Illumina) high- throughput genotyping technology to detect CNVs. In close collaboration with the Department of Statistics at the University of Oxford we developed and validated a novel Bayesian Hidden-Markov Model statistical analysis tool to detect and accurately map CNVs using genotyping data from the BeadArray™ platform. We validated the novel algorithm (QuantiSNP) in the laboratory using a set of well characterized clinical samples and the new analytical tools is being used in several ongoing projects in the centre [16]. This collaboration was extended to the development of other algorithms: GenoSNP for the analysis of genotyping data [20] and OncoSNP for the accurate detection of copy numbers alterations in cancer samples [24].

Other project contributions

As a member of the genomics laboratory I have also been involved in collaborative genotyping projects [13]. In particular I was involved in a project where I shared my transcriptome data analysis skills (using R and Bioconductor Project Packages) to better understand the role of miRNA in hypoxia responses [18, 21]. We had at the time developed a custom microRNA microarrays and I was also involved in a collaboration with Solexa using their new sequencing technology to potentially identify more novel microRNAs. In Oxford I was also involved in training of students and in supervision of research technicians.