Background
Cancers of the vulva are rare gynecological malignancies, primarily affecting elderly women (Herrington, Kim et al. 2020). In 2020, around 45,200 new cases were diagnosed, and 17,400 women died worldwide from these tumors. The most common subtype is squamous cell carcinoma (SCC), accounting for more than 90% of all cancer arising in the vulva, followed by melanoma that accounts for 5-10% of vulvar malignancies. At present, there is no specific target for screening analysis of key molecular genetic events in vulvar carcinogenesis. The rarity of these malignancies is a limiting factor. Despite numerous efforts to investigate these tumors genetically much remains unknown. In short, our molecular genetic information is yet far away from contributing significantly to an early diagnosis and/or successful therapeutic approach to these cancer cases.
Problem statement
The development and progression of cancer is an evolutionary process whereby target cells accumulate multiple mutations at different levels of organization, enabling them to eventually proliferate in an uncontrolled manner, destroy surrounding tissue, and spread to distant locations. To increase existing carcinogenetic knowledge on tumors of the vulva, it is mandatory to start investigating their genome and transcriptome. The following investigations are planned the Section for Cancer Cytogenetics (SCC) and malignant melanoma of the vulva to help overcome the existing ignorance:
- Characterization of the tumors’ molecular landscape at the DNA level:
- Identification of mutations
- Identification of epigenetic changes
- Characterization of the tumors’ transcriptome:
- Gene expression analyses
- miRNA expression investigations
- Identification of fusion genes
Characterization of the molecular landscape, be it identification of mutation(s), fusion gene(s) and/or epigenetic change(s), can help distinguish between driver and passenger events in tumorigenesis. A driver event confers selective advantage onto tumor parenchyma cells and is therefore of fundamental pathogenetic importance. A passenger event involves sequences/genes that do not contribute directly to disease initiation. Their identification and classification may nevertheless contribute diagnostically as well as prognostically. The detection of characteristic biomarker(s) may thus enable better clinical management of the patients inasmuch as they can be stratified into more meaningful subclasses in which treatment is more or less intensive. Eventually, knowledge of gene involvement during tumorigenesis may offer hints as to future targeted therapies.
Research methodology
The general purpose of the research behind this project is to obtain a detailed overview of the genomic and transcriptomic changes harbored by vulvar tumor cells. We are planning to screen tumor genomes in order to detect the presence of mutations and/or alterations in DNA methylation and to investigate tumor transcriptomes searching for fusion transcripts, and to quantify the expression of specific genes and miRNAs known to be relevant in other types of cancers.
Through a solid collaboration with gynecology pathologists, we have, during the past 20 years, collected the largest series of genomically partially characterized such tumors ever reported, something that per se is a treasure trove. The material consists of 85 tumors, of which 45 are SCC, whereas 40 are mucosal melanomas arising in the vagina/vulva. The project has been approved by the Regional Committee for Medical Research Ethics South-East in Norway (REK # 2011/2071).
We plan to use the following methods for the present project:
- Next-Generation Sequencing (NGS of both DNA and RNA)
- DNA methylation analysis
- Gene and miRNA expressions using the nCounter PanCancer Pathway, PanCancer Immune Profiling, and the nCounter Human v3 miRNA Expression Assay Kit.
For all experiments, housekeeping genes and endogenous miRNAs will be incorporated, and positive and negative controls will be used for analysis.
Studentens work plan
The student will be involved in this exciting project by performing specific laboratory tasks as well as building his/her own knowledge about the tumors investigated:
- DNA and RNA extraction from frozen and paraffin-embedded tissues
- DNA and RNA quantification and quality assessment
- DNA library preparation for IonTorrent platform
- Sequencing of the prepared librarie
- RNA hybridization, immobilization, aligning, and counting
- miRNA hybridization, annealing of specific tags to their target miRNAs, ligation reaction, and enzymatic purification
- cDNA synthesis
- primers design for Reverse transcription-polymerase chain reaction (RT–PCR)
- Sequencing and bioinformatic analysis
- Reading literature related to the topic
- Systematic reports of all data obtained
- Presentation of the results and stepwise achievements to the supervisor and the group
About the research environment
The project will be performed at the Section for Cancer Cytogenetics (Radiumhospitalet), which is a leading center for cancer cytogenetic research both nationally and internationally. Our research group specializes in detecting chromosomal and molecular genetic aberrations in cancer and other neoplastic cells. The success we have had in this area can be seen from a large number of publications we have contributed in peer-reviewed scientific journals (more than ten annually in which the first and/or last author comes from our group; 65 during the last five years). The scientific proficiency of our group can also be gleaned from the fact that we have supervised five PhD projects/candidates during 2016-2021, of which four defended their thesis at the Faculty of Medicine and one at the Faculty of Mathematics and Natural Sciences, University of Oslo. We have also supervised exchange students from the ERASMUS program during the same time period. We collaborate nationally and internationally with several groups.
Our group consists of three senior and one young scientist, one consultant oncologist, and two laboratory engineers. We share facilities with the diagnostic part of the Section for Cancer Cytogenetics, whose specialty is to search for chromosomal aberrations in various types of hematological malignancies and solid cancers.
The student will be attached to the gynecological project under the main supervision of Doctor Philos Francesca Micci. He/She will be trained in laboratories techniques and taught the analytical processes by PhD Marta Brunetti, who will act as co-supervisor. Sigbjørn Smeland, Professor at Institute of Clinical Medicine will also act as co-supervisor.
What contributes both diagnostically and in our research is an element in making real so-called “personalized medicine”, also referred to as precision medicine or tailor-made therapy, a medical model that separates patients people based on detailed, relevant information into different groups depending on variations in both host (patient) and invader (tumor) biology. Although tailor-made treatment for patients is a concept that can be traced back to ancient times, the term was used increasingly in recent years based on the new technologies that are now being used to generate an improved understanding of disease biology, especially genomics.