Methodological Approaches towards Personalised Cancer Medicine

Despite advances in diagnostics and treatments, many cancer patients have poor survival rates. Tumours develop drug resistance followed by metastasis, and survivors suffer from treatment side-effects. Omics techniques, targeted treatments and immunotherapy offer the prospect of individually adapting treatments for optimal efficacy and minimal side-effects. This requires integration of biomolecular, clinical and drug data to successfully predict optimal treatments for every patient. The aim of this thesis was to evaluate and apply different methodologies important for personalised treatment in a variety of cancer settings. The first paper showed that E6/E7 mRNA detection through RT-NASBA is more accurate and sensitive than DNA genotyping for classifying HPV infection in cervical adenocarcinoma, showing RNA analysis to be preferable for identifying high-risk patients. In paper II, HPV16 E2 and E5 mRNA expression in oropharyngeal cancer was analysed in relation to clinical outcomes and tumour immunology. Neither down-regulation of HLA class I nor CD8+ T-cell infiltration, both indicators of good prognosis, were dependent on E2 or E5. However, absence of E2 was related to poor progression-free survival. This allows E2 expression to be combined with HLA class I and CD8+ T-cells when stratifying patients with good prognosis for milder treatment. The third paper screened combinations of growth factors and drugs for impact on proliferation of breast cancer cells, creating a two-dimensional space to simulate tumours in different signalling states interacting with drugs. In MDA-MB-231 cells, TGF-β in combination with EGF and oestrogen inhibited growth, with the effect strengthened by Tamoxifen. In MCF7 cells, Tamoxifen inhibited growth when added to both EGF and oestrogen. In paper IV, the immunoproteome in urinary bladder cancer was analysed. Proteomics and network analysis of regulatory (Treg) and effector T-cells (Teff) of lymph nodes showed that Tregs in sentinel nodes (SN) up-regulate growth and immune signalling networks. IL-16, previously not shown to be expressed by Tregs, was predicted as central to SN-Treg signalling. IL-16 expression in Tregs was validated, shown to be higher in lymph nodes than peripheral blood and inhibited by tumour cell supernatant. In conclusion, this thesis has shown methods to improve patient stratification in cervical adenocarcinoma and HPV-positive oropharyngeal cancer. The utility of proliferation screenings replicating tumour heterogeneity for optimising drug combinations was demonstrated, and finally, lymph node proteomics revealed individual differences in T-cell signalling, important for optimising immunotherapy. Integration of these and other methods will be key to arrive at personalised cancer medicine – application of the optimal treatment combination for every patient

Single-cell deconvolution of head and neck squamous cell carcinoma

Complexities in cell-type composition have rightfully led to skepticism and caution in the interpretation of bulk transcriptomic analyses. Recent studies have shown that deconvolution algorithms can be utilized to computationally estimate cell-type proportions from the gene expression data of bulk blood samples, but their performance when applied to tumor tissues, including those from head and neck, remains poorly characterized. Here, we use single-cell data (~6000 single cells) collected from 21 head and neck squamous cell carcinoma (HNSCC) samples to generate cell-type-specific gene expression signatures. We leverage bulk RNA-seq data from \u3e500 HNSCC samples profiled by The Cancer Genome Atlas (TCGA), and using single-cell data as a reference, apply two newly developed deconvolution algorithms (CIBERSORTx and MuSiC) to the bulk transcriptome data to quantitatively estimate cell-type proportions for each tumor in TCGA. We show that these two algorithms produce similar estimates of constituent/major cell-type proportions and that a high T-cell fraction correlates with improved survival. By further characterizing T-cell subpopulations, we identify that regulatory T-cells (

Potential Targets' Analysis Reveals Dual PI3K/mTOR Pathway Inhibition as a Promising Therapeutic Strategy for Uterine Leiomyosarcomas-an ENITEC Group Initiative

Purpose: Uterine sarcomas are rare and heterogeneous tumors characterized by an aggressive clinical behavior. Their high rates of recurrence and mortality point to the urgent need for novel targeted therapies and alternative treatment strategies. However, no molecular prognostic or predictive biomarkers are available so far to guide choice and modality of treatment. Experimental Design: We investigated the expression of several druggable targets (phospho-S6(S240) ribosomal protein, PTEN, PDGFR-alpha, ERBB2, and EGFR) in a large cohort of human uterine sarcoma samples (288), including leiomyosarcomas, low-grade and high-grade endometrial stromal sarcomas, undifferentiated uterine sarcomas, and adenosarcomas, together with 15 smooth muscle tumors of uncertain malignant potential (STUMP), 52 benign uterine stromal tumors, and 41 normal uterine tissues. The potential therapeutic value of the most promising target, p-S6(S240), was tested in patient-derived xenograft (PDX) leiomyosarcoma models. Results: In uterine sarcomas and STUMPs, S6S240 phosphorylation (reflecting mTOR pathway activation) was associated with higher grade (P = 0.001) and recurrence (P = 0.019), as shown by logistic regression. In addition, p-S6(S240) correlated with shorter progression-free survival (P = 0.034). Treatment with a dual PI3K/mTOR inhibitor significantly reduced tumor growth in 4 of 5 leiomyosarcoma PDX models (with tumor shrinkage in 2 models). Remarkably, the 4 responding models showed basal p-S6(S240) expression, whereas the nonresponding model was scored as negative, suggesting a role for p-S6(S240) in response prediction to PI3K/mTOR inhibition. Conclusions: Dual PI3K/mTOR inhibition represents an effective therapeutic strategy in uterine leiomyosarcoma, and p-S6(S240) expression is a potential predictive biomarker for response to treatment. (C)2017 AACR.Peer reviewe

Colorectal cancer incidences in Lynch syndrome: a comparison of results from the prospective lynch syndrome database and the international mismatch repair consortium

Objective To compare colorectal cancer (CRC) incidences in carriers of pathogenic variants of the MMR genes in the PLSD and IMRC cohorts, of which only the former included mandatory colonoscopy surveillance for all participants. Methods CRC incidences were calculated in an intervention group comprising a cohort of confirmed carriers of pathogenic or likely pathogenic variants in mismatch repair genes (path_MMR) followed prospectively by the Prospective Lynch Syndrome Database (PLSD). All had colonoscopy surveillance, with polypectomy when polyps were identified. Comparison was made with a retrospective cohort reported by the International Mismatch Repair Consortium (IMRC). This comprised confirmed and inferred path_MMR carriers who were first- or second-degree relatives of Lynch syndrome probands. Results In the PLSD, 8,153 subjects had follow-up colonoscopy surveillance for a total of 67,604 years and 578 carriers had CRC diagnosed. Average cumulative incidences of CRC in path_MLH1 carriers at 70 years of age were 52% in males and 41% in females; for path_MSH2 50% and 39%; for path_MSH6 13% and 17% and for path_PMS2 11% and 8%. In contrast, in the IMRC cohort, corresponding cumulative incidences were 40% and 27%; 34% and 23%; 16% and 8% and 7% and 6%. Comparing just the European carriers in the two series gave similar findings. Numbers in the PLSD series did not allow comparisons of carriers from other continents separately. Cumulative incidences at 25 years were < 1% in all retrospective groups. Conclusions Prospectively observed CRC incidences (PLSD) in path_MLH1 and path_MSH2 carriers undergoing colonoscopy surveillance and polypectomy were higher than in the retrospective (IMRC) series, and were not reduced in path_MSH6 carriers. These findings were the opposite to those expected. CRC point incidence before 50 years of age was reduced in path_PMS2 carriers subjected to colonoscopy, but not significantly so

Thermal structure of the crust during granulite metamorphism: petrological speculations and geodynamic implications

It is shown on a basis of generalization of lateral and vertical distribution of P-T parameters in granulite-gneiss belts of various ages that the thermal gradient within the lower crust during granulite metamorphism is rather low, ca. 5-10℃/km. Two extreme types of granulitic crustal sections are recognized. First type (GM1) corresponds to normal and especially to thinned (25-40km) crust. High and ultra-high temperatures (up to 1050℃) are characteristic for the deepest granulites of this type. Second type (GM2) corresponds to thickened (50-55km) crust. It is characterized by moderately high temperatures in the lower crust (from 700-800℃ at crust-mantle boundary). GM1 is generally connected with settings related to the hot mantle upwelling to crust-mantle boundary accompanied by heat and penetrating fluid fluxes and crustal extension or crustal thinning. Maximal heating is characteristic for the most thinned crust, i.e., for riftogenic environment including extension zones in back areas of active continental margins and in the crust of epi-continental sedimentary basins. Metamorphism of GM2 type is related to compression conditions and collision crustal stacking

Whole-exome sequencing of HPV positive tonsillar and base of tongue squamous cell carcinomas reveals a global mutational pattern along with relapse-specific somatic variants

To identify predictive/targetable markers in human papillomavirus positive (HPV+) ton-sillar and base of tongue cancer (TSCC/BOTSCC), whole-exome sequencing (WES) of tumours of patients with/without recurrence was performed. Forty primary tumours and adjacent normal tissue were separated by micro-dissection from formalin-fixed paraffin-embedded tissue from patients treated with curative intent 2000–2014 at Karolinska University Hospital. Successful sequencing was obtained in primary tumours of 18 patients without and primaries of 17 with local or distant recurrence, as well as in 10 corresponding recurrences (i.e., five local relapses and five distant metas-tases) from these 17 patients. One variant—a high-impact deletion in the CDC27 gene—was observed only in primaries of 5/17 patients that had a recurrence after full treatment but in none of those without recurrence. In addition, 3 variants and 26 mutated genes, including CDC27, BCLAF1 and AQP7, were present in at least 30% of all primary tumours independent of prognosis. To conclude, a CDC27 deletion was specific and found in ~30% of samples from patients with a local relapse/distant metastasis and could, therefore, potentially be a prospective marker to predict prognosis. Commonly mutated genes, such as BCLAF1, should be further studied in the context of targeted therapy