Research team Tina Dalianis
Studies on human papillomaviruses and specific markers for tailored and targeted therapy of head and neck cancer and childhood cancer.
Studies on head and neck cancer
Background and previous results
There are >150 HPV types of which some types or species are potentially oncogenic. Best known are HPV16 and 18, which cause cervical cancer and included in today's HPV vaccines. However, besides smoking and alcohol, HPV16 has a causal link to oropharyngeal cancer, where tonsillar and base of tongue cancer (TSCC and BOTSCC) dominate. We and others have shown that HPV-positive TSCC and BOTSCC have a much better survival than corresponding HPV-negative cancers and head/neck cancers in general (80% vs. 40% 5-year disease-specific survival). In addition, we were the first to show that the incidences of HPV-positive TSCC and BOTSCC have increased epidemically the past decades. Due to its poor prognosis, therapy for head and neck squamous cell carcinoma (HNSCC) generally includes chemotherapy and intensified radiotherapy (RT), with many more severe side effects. Intensified therapy has not improved survival for those with a poor prognosis and some patients are likely overtreated. Clearly therefore, new therapeutic options are needed for this group of patients that today account for >40% of all HNSCC. However, to better individualize therapy it is important to find markers that along with HPV can better predict a favorable response to conventional treatment, or that can be useful for targeted therapy.
Previous data and research plan
In a TSCC and BOTSCC patient cohort from 2000-2011 at the Karolinska University Hospital, we identified most but not all such markers using immunohistochemistry (e.g. HLA class I, CD44, LRIG, CD8 tumour-infiltrating lymphocyte counts, expression of HPV16 E2 mRNA), which individually or together identify 20-30% of patients with 90-95% probability for complete response. Using mathematical models we later combined HPV-positive status with several prognostic biomarkers and can identify 40-56% of the patients that respond to therapy.
We then proceeded to find additional markers using DNA and RNA sequencing, some data are described below.
Examining hotspot mutations by Next Generation Sequencing of 50 cancer related genes in 300 HPV+ TSCC/BOTSCC and 50 HPV- TSCC/BOTSCC and found that the genes PIK3CA and FGFR3, both targetable proteins, were among the most mutated ones in HPV+ TSCC/BOTSCC, while p53 was most frequently mutated in HPV-TSCC/BOTSCC (Fig.1). Notably, we also found that mutated FGFR3 in HPV positive tumours correlated to worse prognosis. Furthermore, others have also reported that when de-escalating treatment patients with tumours with PIK3CA mutations did worse.
The above data indicated that targeting PI3K and FGFR3 could potentially be useful for this patient group.
When performing protein profiling done by a sensitive proximity elongation assay (PEA) we found that some proteins related to angiogenesis, e.g. VEGF-A, CYR61 and ESM-1 and IGF1R, the latter conferring resistance to EGFR inhibitors, are up-regulated, while hK-8 is low, in HPV+ TSCC/BOTSCC that do not respond to treatment, indicating that these proteins can be used to predict effect of therapy.
More recently, we performed whole exome sequencing (WES) on tumors of patients with and without relapse and found some genes, e.g. a specific CDC27 deletion variant that was present only in patients that had encountered a relapse, while others such as BLAF1 and AQP1 as well as CDC27 had mutations in their genes both cohorts. Hopefully some of the data here will contribute to finding new options for targeted therapy.
To acquire more data on targeted therapy, we have employed in vitro models tissue culture models for targeted therapy. HPV positive and HPV negative TSCC and BOTSCC cell lines with or without e.g. FGFR3 and PIK3CA mutations have been obtained. These TSCC/BOTSCC cell lines and have been tested for their sensitivity to different targeted therapies using inhibitors against PI3K, FGFR, CDK4/6, PARP and WEE1 inhibitors by viability, proliferation, apoptosis, cytotoxicity and FACs assays.
These experiments that have been performed were very promising. We have for example found that our cell lines show dose dependent responses to most of the inhibitors. However, when some of these inhibitors are combined, both additive and synergistic effects were disclosed. We are now combining these inhibitors with irradiation or chemotherapy and studying their joint effects.
With this approach we hope to identify whether drugs used today (indicated above) for targeted therapy in other types of tumors, or against newly disclosed mutated genes by WES also can be used for TSCC and BOTSCC. Useful drugs will also be further analyzed in experimental animal PDX systems.
Moreover, we are presently barcoding our cell lines in order to follow subclonal evolution after various drug treatment and performing single DNA and RNA sequencing to study resistant patterns according to the specific molecular profile of each tumour cell line.
Finally, we have just initiated some additional studies on HPV viral load and cell free HPV DNA in order to follow prognosis and possible risk for relapse.
Studies of childhood cancer
In parallel with our studies above, we have also screened a number of childhood cancers such as a number of different childhood brain tumors and neuroblastomas for the presence of FGFR3 and PIK3CA mutations. These types of mutations are rare, but have been found in at least one neuroblastoma (NB). We are therefore also testing some NB cell lines with the same assays that have been described above for the tonsillar and base of tongue cancer (TSCC and BOTSCC) cell lines. We have found some positive effects and these are investigated further by team Ourania Kostopoulou in our group.
Using novel tools to better prognosticate and tailor therapy for growing numbers of TSCC/BOTSCC patients, it will be possible offer targeted therapy for those with poor prognosis and avoid overtreatment of those with good prognosis, which will thereby increase survival as well as quality of life.
For childhood cancer, if targeted therapy can be used synergistically this may be useful for those with tumors that are resistant to conventional chemotherapy and help increase survival as well as avoid some side effects associated with conventional chemotherapy.
Tina Dalianis, Professor em. Group leader
Torbjörn Ramqvist, Assistant Professor, associated
Stefan Holzhauser, PhD, Post doc
Ourania Kostopoulou, PhD, Associate Professor
Mark Zupancic, MD, PhD, post doc
Juan Du, PhD, Post doc, associated
Michael Mints MD, PhD, Post doc, associated
Andrea Vlastos, MD, PhD, ENT specialist, associated
Filippo Tuti, MSci, KTH
Karin Byskata, BSci student, KI