< Terug naar vorige pagina
Publicatie
In situ MRNA vaccination
Boek - Dissertatie
Ondertitel:bringing the fight to the tumor
Breast cancer (BC) is the most frequently diagnosed cancer in women worldwide. Current treatment modalities range from surgery, chemotherapy and radiotherapy to targeted therapy and immunotherapy.
Recurrence of BC cells often occurs. Therefore, new treatment options that are minimally invasive and activate memory T-cells are urgently needed. As BC is a heterogeneous disease, there is also a need to (molecularly) portrait the disease to stratify patients for precision therapy.
The density, composition, functional state, and organization of immune components in BC tumors provides predictive and prognostic information that helps guide therapy-decision making. Tumor-infiltrating immune cells are of prognostic and predictive value in human epidermal growth factor receptor 2 (HER2)+ BC and triple-negative breast cancer (TNBC). We probed analysis of infiltrating immune cells using immunohistochemistry (IHC) and NanoString gene expression profiling (GEP) to ascertain their value to monitor the immune response in a phase I clinical trial in BC patients treated with an intralesional injection of TriMix mRNA (NCT03788083). Immune cells identified by IHC are further referred to as stromal tumor-infiltrating lymphocytes (sTILs), while those identified using GEP also comprise other populations than lymphocytes and are therefore referred to a tumor-infiltratingleukocytes (TILs). Prior to this work, we optimized and validated an RNA-extraction protocol from freshfrozen (FF) and formalin-fixed paraffin-embedded (FFPE) core needle biopsies (CNBs). RNA extraction from FF CNBs requires mechanical disruption and resulted in 92% of the cases in sufficient yield and quality RNA. For the remaining 8%, FFPE specimens that are routinely generated for histopathology and diagnostic purposes, can serve as a back-up plan. Using a protocol for microtome sectioning in an RNase free manner, we extracted lower, but sufficient amounts of lower quality RNA compared to FF CNBs. However, RNA extracted from as well as FF as FFPE CNB specimens were compatible with NanoString Technologies.
Next, we showed that sTIL-scoring by IHC on hematoxylin and eosin (HE) stained tissue sections was prone to intra- and inter-rater variability, which could be reduced by staining tissue sections with antibodies that identify T-cells (CD3) or cytotoxic T lymphocytes (CD8). The sTIL-scores obtained after additional immune cell staining correlated well with the TIL-scores obtained via NanoString GEP. Gene expression data were further scrutinized against an existing single-cell RNA sequencing (scRNAseq) dataset. We showed that GEP in BC tumors with a high (s)TIL-score identified classically activated myeloid cells, while GEP in BC tumors with a low (s)TIL-score identified regulatory T cells (Tregs) and immunosuppressive myeloid cells. These findings confirm the value of sTILs as a biomarker in BC and justify the use of IHC and NanoString GEP to refine and complement sTIL-scoring.
The optimized workflow consisting of sTIL-scoring on tissue sections versus TIL-scoring and extended gene expression analysis on RNA was used to analyze the outcome of intralesional injection of increasing doses of TriMix mRNA compared to placebo. At the time of writing this thesis, 4 patients were included in the placebo cohort, while 3 patients were included in the dose level I (1 mg/mL TriMix mRNA) and dose level II (3 mg/mL TriMix mRNA) treatment cohorts. The rationale of the study was that TriMix mRNA, consisting of mixture of three mRNA molecules encoding CD40 Ligand (CD40L), CD70 and a constitutively active toll-like receptor 4 (caTLR4), can be engulfed by dendritic cells (DCs) within the tumor enabling them to activate T cells against the tumor antigens expressed by the BC cells.
Intratumoral administration of TriMix mRNA was safe and tolerable, inducing only mild, manageable and reversible adverse events (AEs). Three out of six patients treated with TriMix mRNA showed a decrease in the peripheral blood neutrophil to lymphocyte ratio. Spatial and quantitative sTIL-levels were assessed in pre- and post-treatment BC tumors, showing an increase in CD3+ sTILs according to IHC analysis and subtle changes at the gene expression level, hinting towards a T-cell response in the patient cohort treated with 3mg/mL TriMix mRNA.
In conclusion, we contend that the tumor immune contexture can be used to estimate cancer prognosis, therapy management and response. We have shown that the proposed technologies, IHC and NanoString GEP can help unravel the cellular and transcriptomic components of the tumor microenvironment (TME), thereby aiding in effective, wisely timed, rational therapy-decision making.
Recurrence of BC cells often occurs. Therefore, new treatment options that are minimally invasive and activate memory T-cells are urgently needed. As BC is a heterogeneous disease, there is also a need to (molecularly) portrait the disease to stratify patients for precision therapy.
The density, composition, functional state, and organization of immune components in BC tumors provides predictive and prognostic information that helps guide therapy-decision making. Tumor-infiltrating immune cells are of prognostic and predictive value in human epidermal growth factor receptor 2 (HER2)+ BC and triple-negative breast cancer (TNBC). We probed analysis of infiltrating immune cells using immunohistochemistry (IHC) and NanoString gene expression profiling (GEP) to ascertain their value to monitor the immune response in a phase I clinical trial in BC patients treated with an intralesional injection of TriMix mRNA (NCT03788083). Immune cells identified by IHC are further referred to as stromal tumor-infiltrating lymphocytes (sTILs), while those identified using GEP also comprise other populations than lymphocytes and are therefore referred to a tumor-infiltratingleukocytes (TILs). Prior to this work, we optimized and validated an RNA-extraction protocol from freshfrozen (FF) and formalin-fixed paraffin-embedded (FFPE) core needle biopsies (CNBs). RNA extraction from FF CNBs requires mechanical disruption and resulted in 92% of the cases in sufficient yield and quality RNA. For the remaining 8%, FFPE specimens that are routinely generated for histopathology and diagnostic purposes, can serve as a back-up plan. Using a protocol for microtome sectioning in an RNase free manner, we extracted lower, but sufficient amounts of lower quality RNA compared to FF CNBs. However, RNA extracted from as well as FF as FFPE CNB specimens were compatible with NanoString Technologies.
Next, we showed that sTIL-scoring by IHC on hematoxylin and eosin (HE) stained tissue sections was prone to intra- and inter-rater variability, which could be reduced by staining tissue sections with antibodies that identify T-cells (CD3) or cytotoxic T lymphocytes (CD8). The sTIL-scores obtained after additional immune cell staining correlated well with the TIL-scores obtained via NanoString GEP. Gene expression data were further scrutinized against an existing single-cell RNA sequencing (scRNAseq) dataset. We showed that GEP in BC tumors with a high (s)TIL-score identified classically activated myeloid cells, while GEP in BC tumors with a low (s)TIL-score identified regulatory T cells (Tregs) and immunosuppressive myeloid cells. These findings confirm the value of sTILs as a biomarker in BC and justify the use of IHC and NanoString GEP to refine and complement sTIL-scoring.
The optimized workflow consisting of sTIL-scoring on tissue sections versus TIL-scoring and extended gene expression analysis on RNA was used to analyze the outcome of intralesional injection of increasing doses of TriMix mRNA compared to placebo. At the time of writing this thesis, 4 patients were included in the placebo cohort, while 3 patients were included in the dose level I (1 mg/mL TriMix mRNA) and dose level II (3 mg/mL TriMix mRNA) treatment cohorts. The rationale of the study was that TriMix mRNA, consisting of mixture of three mRNA molecules encoding CD40 Ligand (CD40L), CD70 and a constitutively active toll-like receptor 4 (caTLR4), can be engulfed by dendritic cells (DCs) within the tumor enabling them to activate T cells against the tumor antigens expressed by the BC cells.
Intratumoral administration of TriMix mRNA was safe and tolerable, inducing only mild, manageable and reversible adverse events (AEs). Three out of six patients treated with TriMix mRNA showed a decrease in the peripheral blood neutrophil to lymphocyte ratio. Spatial and quantitative sTIL-levels were assessed in pre- and post-treatment BC tumors, showing an increase in CD3+ sTILs according to IHC analysis and subtle changes at the gene expression level, hinting towards a T-cell response in the patient cohort treated with 3mg/mL TriMix mRNA.
In conclusion, we contend that the tumor immune contexture can be used to estimate cancer prognosis, therapy management and response. We have shown that the proposed technologies, IHC and NanoString GEP can help unravel the cellular and transcriptomic components of the tumor microenvironment (TME), thereby aiding in effective, wisely timed, rational therapy-decision making.
Aantal pagina's: 184
Jaar van publicatie:2022
Trefwoorden:mRNA vaccination, tumor, cancer, treatment
Toegankelijkheid:Open