| 1. |
- Backman, Max, 1987-
(författare)
-
Spatial immune analyses in clinical cancer tissue
- 2022
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cancer is a leading cause of premature death and lung cancer is the deadliest cancer type, with non-small cell lung cancer (NSCLC) representing 85% of lung cancer cases. Despite promising development in cancer treatment in recent decades, overall prognosis is poor. The aim of this thesis was to explore novel techniques in protein visualization in clinical cancer tissue to better our understanding of cancer immunity and to discover new biomarkers for improved cancer diagnostics.In Paper I traditional immunohistochemistry (IHC) was compared to the in-situ proximity ligation assay (isPLA). Both techniques were applied to stain 12 proteins in 39 cell lines and 37 tissue types. Two different antibodies were used in the IHC assay and in the isPLA, where binding by both antibodies is required to generate detection signals. The comparison of staining patterns showed that the isPLA presents a valuable alternative to traditional IHC.In Paper II cancer tissue from 357 NSCLC patients was immunophenotyped through IHC annotations of 11 different immune markers. A distinct group of cases with a signature of NK cells and/or plasma cells had favorable prognosis despite significantly lower T-cell activation signatures. This study provides a detailed description of the immune landscape in NSCLC, extending previous concepts, and highlights plasma and NK-cells as potential biomarkers for further validation.In Paper III a multiplex-multispectral pipeline was established to explore three immune marker panels in a NSCLC cohort, spatially quantifying 13 immune cell types. The immune composition of NSCLC was analyzed for the prognostic relevance of immune cell coordination. Cell densities and distances were found to contribute independently to prognosis, indicating that spatial information on local immune cell infiltration is crucial for understanding tumor immunity.In Paper IV an extensive characterization of the immune cell landscape of colon cancer identified a prognostic signature based on the ratio of CD8+ lymphocytes to CD68+CD163+ macrophages. This signature was superior to the state-of-the-art ‘Immunoscore’, and was also associated with longer survival when analyzed in other common cancer types. This presents a promising immunological biomarker that warrants further validation as a prognostic and predictive signature in common cancers.In summary, this thesis presents an in-depth study of immune cell infiltration in several cancer types to better understand cancer immunity. Through novel techniques and spatial metrics, we describe immunophenotypes that might contribute to cancer classification and prognostication. The identified immune phenomena may also present alternative treatment targets to overcome resistance to immunotherapy.
|
|
| 2. |
- Elfving, Hedvig
(författare)
-
Improving the diagnostic armamentarium of lung cancer
- 2024
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Lung cancer is the leading cause of cancer-related death globally, with non-small cell lung cancer (NSCLC) constituting 85% of cases. The introduction of immunotherapies and targeted therapies have dramatically improved the prognosis and outcome for a subset of patients, and stressed the development of diagnostic tools for effective patient selection. This thesis addresses different aspects of current and future diagnostic strategies in NSCLC patients.In Paper I, an immunohistochemical assay targeting the neurotropic tropomyosin receptor kinase (NTRK) was evaluated on tissue microarrays (TMAs) from a well-characterized NSCLC cohort. Although a few cases showed positive staining, none were positive in the reference molecular testing, highlighting the rarity of this targetable aberration and underscoring the value of cost-effective screening methods.In Paper II, paired patient samples (biopsy, TMA, and surgical specimen) were evaluated regarding the immunohistochemical staining for PD-L1. The results indicated a strong correlation between the PD-L1 expression on biopsy and TMA compared with the tumor whole slide, suggesting that tumor heterogeneity has minor relevance for PD-L1 evaluation.In Paper III, paired tissue samples (biopsy, TMA, and surgical specimen) were evaluated regarding infiltrating CD3+ immune cells. Only weak correlation was found between the biopsies and tumor whole slide, while the agreement between the whole slide and TMA was higher. These results question the use of biopsies for immune cell quantification, while supporting the TMA as a reliable tool in cancer research.In Paper IV, the amount and distribution of tertiary lymphoid structures (TLS) was annotated on scanned tumor whole slides. TLS were present in most of the tumors and correlated with the abundance of specific immune cell subsets. Higher number of TLS were associated with longer survival, particularly in adenocarcinomas. High tumor mutational burden was associated with higher numbers of periphery TLS. These results provide a rationale for using TLS metrics as a diagnostic marker for NSCLC patients undergoing surgical resection.In summary, this thesis addresses challenges in prognostic and predictive lung cancer diagnostics in the areas of targeted therapy and immunotherapy. The results provide crucial insights into tumor heterogeneity that may impact clinical decision-making and aid scientists in selecting appropriate tissue sources for translational and clinical cancer research.
|
|
| 3. |
- Backman, Max, et al.
(författare)
-
Infiltration of NK and plasma cells is associated with a distinct immune subset in non‐small cell lung cancer
- 2021
-
Ingår i: Journal of Pathology. - : John Wiley & Sons. - 0022-3417 .- 1096-9896. ; 255:3, s. 243-256
-
Tidskriftsartikel (refereegranskat)abstract
- Immune cells of the tumor microenvironment are central but erratic targets for immunotherapy. The aim of this study was to characterize novel patterns of immune cell infiltration in non-small cell lung cancer (NSCLC) in relation to its molecular and clinicopathologic characteristics. Lymphocytes (CD3+, CD4+, CD8+, CD20+, FOXP3+, CD45RO+), macrophages (CD163+), plasma cells (CD138+), NK cells (NKp46+), PD1+, and PD-L1+ were annotated on a tissue microarray including 357 NSCLC cases. Somatic mutations were analyzed by targeted sequencing for 82 genes and a tumor mutational load score was estimated. Transcriptomic immune patterns were established in 197 patients based on RNA sequencing data. The immune cell infiltration was variable and showed only poor association with specific mutations. The previously defined immune phenotypic patterns, desert, inflamed, and immune excluded, comprised 30, 13, and 57% of cases, respectively. Notably, mRNA immune activation and high estimated tumor mutational load were unique only for the inflamed pattern. However, in the unsupervised cluster analysis, including all immune cell markers, these conceptual patterns were only weakly reproduced. Instead, four immune classes were identified: (1) high immune cell infiltration, (2) high immune cell infiltration with abundance of CD20+ B cells, (3) low immune cell infiltration, and (4) a phenotype with an imprint of plasma cells and NK cells. This latter class was linked to better survival despite exhibiting low expression of immune response-related genes (e.g. CXCL9, GZMB, INFG, CTLA4). This compartment-specific immune cell analysis in the context of the molecular and clinical background of NSCLC reveals two previously unrecognized immune classes. A refined immune classification, including traits of the humoral and innate immune response, is important to define the immunogenic potency of NSCLC in the era of immunotherapy. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
|
|
| 4. |
- Backman, Max, 1987-, et al.
(författare)
-
Spatial immunophenotyping of the tumour microenvironment in non-small cell lung cancer
- 2023
-
Ingår i: European Journal of Cancer. - : Elsevier. - 0959-8049 .- 1879-0852. ; 185, s. 40-52
-
Tidskriftsartikel (refereegranskat)abstract
- Introduction: Immune cells in the tumour microenvironment are associated with prognosis and response to therapy. We aimed to comprehensively characterise the spatial im-mune phenotypes in the mutational and clinicopathological background of non-small cell lung cancer (NSCLC).Methods: We established a multiplexed fluorescence imaging pipeline to spatially quantify 13 immune cell subsets in 359 NSCLC cases: CD4 effector cells (CD4-Eff), CD4 regulatory cells (CD4-Treg), CD8 effector cells (CD8-Eff), CD8 regulatory cells (CD8-Treg), B-cells, natural killer cells, natural killer T-cells, M1 macrophages (M1), CD163 thorn myeloid cells (CD163), M2 macrophages (M2), immature dendritic cells (iDCs), mature dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs).Results: CD4-Eff cells, CD8-Eff cells and M1 macrophages were the most abundant immune cells invading the tumour cell compartment and indicated a patient group with a favourable prognosis in the cluster analysis. Likewise, single densities of lymphocytic subsets (CD4-Eff, CD4-Treg, CD8-Treg, B-cells and pDCs) were independently associated with longer survival. However, when these immune cells were located close to CD8-Treg cells, the favourable impact was attenuated. In the multivariable Cox regression model, including cell densities and distances, the densities of M1 and CD163 cells and distances between cells (CD8-Treg-B-cells, CD8-Eff-cancer cells and B-cells-CD4-Treg) demonstrated positive prognostic impact, whereas short M2-M1 distances were prognostically unfavourable.Conclusion: We present a unique spatial profile of the in situ immune cell landscape in NSCLC as a publicly available data set. Cell densities and cell distances contribute independently to prognostic information on clinical outcomes, suggesting that spatial information is crucial for diagnostic use.
|
|
| 5. |
- Djureinovic, Dijana
(författare)
-
Transcriptomic and Proteomic Analysis of Tumor Markers in Tissue and Blood from Patients with Lung Cancer
- 2018
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Despite recent treatment advancements, the survival outcome remains poor for the majority of patients with non-small cell lung cancer (NSCLC). The aim of this thesis was to evaluate protein expression to predict prognosis and identify biomarkers that can be used as targets for immunotherapy or for early detection of NSCLC.In Paper I an optimized immunohistochemistry (IHC)-based prognostic model was developed for NSCLC. The prognostic performance of the model was compared to the clinicopathological parameters that are used in the clinical setting to predict outcome. The protein model failed to outperform clinicopathological parameters in predicting survival outcome questioning the potential of IHC-based assessment of prognostic markers in NSCLC.In Paper II the human testis-specific proteome was profiled using RNA-sequencing (RNA-seq) data from testis and 26 other organs. More than 1000 genes demonstrated a testis-enriched expression pattern which makes testis the tissue with the most tissue-specific genes. The majority of the testis-enriched genes were previously poorly described and were further profiled by IHC. This analysis provides a starting point to increase the molecular understanding of testicular biology.In Paper III the profiling of cancer-testis antigens (CTAs) was performed in NSCLC by using RNA-seq data from 32 normal organs and NSCLC. Ninety genes showed CTA expression profiles. The transcriptomic data were validated by IHC for several CTAs. The comprehensive analysis of CTAs can guide biomarker studies or help to identify targets for immunotherapeutic strategies.In Paper IV the reactivity of CTAs was evaluated by measuring the abundance of autoantibodies in plasma from patients with NSCLC and benign lung diseases. Twenty-nine CTAs demonstrated exclusive reactivity in NSCLC and six of them were reactive in an independent NSCLC cohort. These findings suggest that some CTAs are immunogenic and could be utilized in immunotherapy.In Paper V an immunoassay was used on lung adenocarcinoma plasma samples and samples from benign lung diseases. The plasma levels of 92 cancer related proteins were used to build a model that discriminated lung adenocarcinoma from benign controls with a sensitivity of 93% and a specificity of 64%. The results indicate that this assay is promising for the early detection of NSCLC.In summary, this thesis presents an integrative analysis of lung cancer tissue and blood samples to characterize NSCLC on the transcriptomic and proteomic level and to identify cancer specific proteins.
|
|
| 6. |
- Elfving, Hedvig, et al.
(författare)
-
Spatial distribution of tertiary lymphoid structures in the molecular and clinical context of non-small cell lung cancer.
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Tertiary lymphoid structures (TLS) are lymphocyte aggregates resembling secondary lymphoid organs and are pivotal in cancer immunity. The ambiguous morphological definition of TLS makes it challenging to ascertain their clinical impact on patient survival and response to immunotherapy. This study aimed to characterize TLS in hematoxylin-eosin tissue sections from lung cancer patients, assessing their occurrence in relation to the local immune environment, mutational background, and patient outcome.Two pathologists evaluated one whole tissue section from each resection specimen of 680 NSCLC patients. TLS were spatially quantified within the tumor area or periphery and further categorized based on the presence of germinal centers (mature TLS). Metrics were integrated with immune cell counts, genomic and transcriptomic data, and correlated with clinical parameters.Out of 536 evaluable cases, TLS were present in 86% of tumor samples, predominantly in the tumor periphery, with a median of eight TLS per case. TLS with germinal centers were found in 24% of cases. TLS presence correlated positively with increased plasma cell (CD138+) and lymphocytic cell (CD3+, CD8+, FOXP3+) infiltration. Tumors with higher tumor mutational burden (TMB) exhibited higher periphery TLS numbers. The overall TLS quantity was associated with improved patient survival, irrespective of TLS maturation status. This prognostic association held true for periphery TLS but not for tumor TLS.In conclusion, TLS occurrence in NSCLC is common and its correlation with a specific immune phenotype suggests biological relevance in the local immune reaction. The prognostic significance of this scoring system on routine hematoxylin-eosin sections has the potential to augment diagnostic algorithms for NSCLC patients.
|
|
| 7. |
- Ericson Lindquist, Kajsa, et al.
(författare)
-
Difficulties in diagnostics of lung tumours in biopsies : an interpathologist concordance study evaluating the international diagnostic guidelines
- 2022
-
Ingår i: Journal of Clinical Pathology. - : BMJ Publishing Group Ltd. - 0021-9746 .- 1472-4146. ; 75:5, s. 302-309
-
Tidskriftsartikel (refereegranskat)abstract
- AIMS: Accurate and reliable diagnosis is essential for lung cancer treatment. The study aim was to investigate interpathologist diagnostic concordance for pulmonary tumours according to WHO diagnostic criteria.METHODS: Fifty-two unselected lung and bronchial biopsies were diagnosed by a thoracic pathologist based on a broad spectrum of immunohistochemical (IHC) stainings, molecular data and clinical/radiological information. Slides stained with H&E, thyroid transcription factor-1 (TTF-1) clone SPT24 and p40 were scanned and provided digitally to 20 pathologists unaware of reference diagnoses. The pathologists independently diagnosed the cases and stated if further diagnostic markers were deemed necessary.RESULTS: In 31 (60%) of the cases, ≥80% of the pathologists agreed with each other and with the reference diagnosis. Lower agreement was seen in non-small cell neuroendocrine tumours and in squamous cell carcinoma with diffuse TTF-1 positivity. Agreement with the reference diagnosis ranged from 26 to 45 (50%-87%) for the individual pathologists. The pathologists requested additional IHC staining in 15-44 (29%-85%) of the 52 cases. In nearly half (17 of 36) of the malignant cases, one or more pathologist advocated for a different final diagnosis than the reference without need of additional IHC markers, potentially leading to different clinical treatment.CONCLUSIONS: Interpathologist diagnostic agreement is moderate for small unselected bronchial and lung biopsies based on a minimal panel of markers. Neuroendocrine morphology is sometimes missed and TTF-1 clone SPT24 should be interpreted with caution. Our results suggest an intensified education need for thoracic pathologists and a more generous use of diagnostic IHC markers.
|
|
| 8. |
- Thurfjell, Viktoria, et al.
(författare)
-
Comparison of ROS1-rearrangement detection methods in a cohort of surgically resected non-small cell lung carcinomas
- 2022
-
Ingår i: Translational Lung Cancer Research (TLCR). - : AME Publishing. - 2218-6751 .- 2226-4477. ; 11:12, s. 2477-2494
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Patients with non-small cell lung cancer (NSCLC) harboring a ROS proto-oncogene 1 (ROS1)-rearrangement respond to treatment with ROS1 inhibitors. To distinguish these rare cases, screening with immunohistochemistry (IHC) for ROS1 protein expression has been suggested. However, the reliability of such an assay and the comparability of the antibody clones has been debated. Therefore we evaluated the diagnostic performance of current detection strategies for ROS1-rearrangement in two NSCLC-patient cohorts.Methods: Resected tissue samples, retrospectively collected from consecutive NSCLC-patients surgically treated at Uppsala University Hospital were incorporated into tissue microarrays [all n=676, adenocarcinomas (AC) n=40 1, squamous cell carcinomas (SCC) n=2 13, other NSCLC n=62]. ROS1rearrangements were detected using fluorescence in situ hybridization (FISH) (Abbott Molecular; ZytoVision). In parallel, ROS1 protein expression was detected using IHC with three antibody clones (D4D6, SP384, EPMGHR2) and accuracy, sensitivity, and specificity were determined. Gene expression microarray data (Affymetrix) and RNA-sequencing data were available for a subset of patients. NanoString analyses were performed for samples with positive or ambiguous results (n=21).Results: Using FISH, 2/630 (0.3% all NSCLC; 0.5% non-squamous NSCLC) cases were positive for ROS1 fusion. Additionally, nine cases demonstrated ambiguous FISH results. Using IHC, ROS1 protein expression was detected in 24/665 (3.6% all NSCLC; 5.1% non-squamous NSCLC) cases with clone D4D6, in 18/639 (2.8% all NSCLC; 3.9% non-squamous NSCLC) cases with clone SP384, and in 1/593 (0.2% all NSCLC; 0.3% non-squamous NSCLC) case with clone EPMGHR2. Elevated RNA-levels were seen in 19/369 (5.1%) cases (Affymetrix and RNA-sequencing combined). The overlap of positive results between the assays was poor. Only one of the FISH-positive cases was positive with all antibodies and demonstrated high RNA-expression. This rearrangement was confirmed in the NanoString-assay and also in the RNA sequencing data. Other cases with high protein/RNA-expression or ambiguous FISH were negative in the NanoString-assay.Conclusions: The occurrence of ROS1 fusions is low in our cohorts. The IHC assays detected the fusions, but the accuracy varied depending on the clone. The presumably false-positive and uncertain FISH results questions this method for detection of ROS1-rearrangements. Thus, when IHC is used for screening, transcript-based assays are preferable for validation in clinical diagnostics.
|
|
