Use of beta-blockers in patients with ductal carcinoma in situ and risk of invasive breast cancer recurrence: a Swedish retrospective cohort study.

BACKGROUND: Retrospective observational studies suggest a potential role of beta-blockers as a protective strategy against progression and metastasis in invasive breast cancer. In this context, we investigated the impact of beta-blocker exposure on risk for progression to invasive breast cancer after diagnosis of ductal cancer in situ (DCIS). METHODS: The retrospective study population included 2535 women diagnosed with pure DCIS between 2006 and2012 in three healthcare regions in SwedenExposure to beta-blocker was quantified using a time-varying percentage of days with medication available. The absolute risk was quantified using cumulative incidence functions and cox models were applied to quantify the association between beta-blocker exposure and time from DCIS diagnosis to invasive breast cancer, accounting for delayed effects, competing risks and pre-specified confounders. RESULTS: The median follow-up was 8.7 years. One third of the patients in our cohort were exposed to beta-blockers post DCIS diagnosis. During the study period, 48 patients experienced an invasive recurrence, giving a cumulative incidence of invasive breast cancer progression of 1.8% at five years. The cumulative exposure to beta-blocker was associated with a reduced risk in a dose-dependent manner, though the effect was not statistically significant. CONCLUSION: Our observational study is suggestive of a protective effect of beta-blockers against invasive breast cancer after primary DCIS diagnosis. These results provide rationales for experimental and clinical follow-up studies in carefully selected DCIS groups.

Tumor Heterogeneity Confounds Lymphocyte Metrics in Diagnostic Lung Cancer Biopsies.

CONTEXT.­: The immune microenvironment is involved in fundamental aspects of tumorigenesis, and immune scores are now being developed for clinical diagnostics. OBJECTIVE.­: To evaluate how well small diagnostic biopsies and tissue microarrays (TMAs) reflect immune cell infiltration compared to the whole tumor slide, in tissue from patients with non-small cell lung cancer. DESIGN.­: A TMA was constructed comprising tissue from surgical resection specimens of 58 patients with non-small cell lung cancer, with available preoperative biopsy material. Whole sections, biopsies, and TMA were stained for the pan-T lymphocyte marker CD3 to determine densities of tumor-infiltrating lymphocytes. Immune cell infiltration was assessed semiquantitatively as well as objectively with a microscopic grid count. For 19 of the cases, RNA sequencing data were available. RESULTS.­: The semiquantitative comparison of immune cell infiltration between the whole section and the biopsy displayed fair agreement (intraclass correlation coefficient [ICC], 0.29; P = .01; CI, 0.03-0.51). In contrast, the TMA showed substantial agreement compared with the whole slide (ICC, 0.64; P < .001; CI, 0.39-0.79). The grid-based method did not enhance the agreement between the different tissue materials. The comparison of CD3 RNA sequencing data with CD3 cell annotations confirmed the poor representativity of biopsies as well as the stronger correlation for the TMA cores. CONCLUSIONS.­: Although overall lymphocyte infiltration is relatively well represented on TMAs, the representativity in diagnostic lung cancer biopsies is poor. This finding challenges the concept of using biopsies to establish immune scores as prognostic or predictive biomarkers for diagnostic applications.

An idiosyncratic zonated stroma encapsulates desmoplastic liver metastases and originates from injured liver.

A perimetastatic capsule is a strong positive prognostic factor in liver metastases, but its origin remains unclear. Here, we systematically quantify the capsule's extent and cellular composition in 263 patients with colorectal cancer liver metastases to investigate its clinical significance and origin. We show that survival improves proportionally with increasing encapsulation and decreasing tumor-hepatocyte contact. Immunostaining reveals the gradual zonation of the capsule, transitioning from benign-like NGFRhigh stroma at the liver edge to FAPhigh stroma towards the tumor. Encapsulation correlates with decreased tumor viability and preoperative chemotherapy. In mice, chemotherapy and tumor cell ablation induce capsule formation. Our results suggest that encapsulation develops where tumor invasion into the liver plates stalls, representing a reparative process rather than tumor-induced desmoplasia. We propose a model of metastases growth, where the efficient tumor colonization of the liver parenchyma and a reparative liver injury reaction are opposing determinants of metastasis aggressiveness.

Vessel size as a marker of survival in estrogen receptor positive breast cancer.

PURPOSE: Angiogenesis is crucial for tumor growth and is one of the hallmarks of cancer. In this study, we analyzed microvessel density, vessel median size, and perivascular a-SMA expression as prognostic biomarkers in breast cancer. METHODS: Dual IHC staining was performed where alpha-SMA antibodies were used together with antibodies against the endothelial cell marker CD34. Digital images of stainings were analyzed to extract quantitative data on vessel density, vessel size, and perivascular alpha-SMA status. RESULTS: The analyses in the discovery cohort (n = 108) revealed a statistically significant relationship between large vessel size and shorter disease-specific survival (p = 0.007, log-rank test; p = 0.01, HR 3.1; 95% CI 1.3-7.4, Cox-regression analyses). Subset analyses indicated that the survival association of vessel size was strengthened in ER + breast cancer. To consolidate these findings, additional analyses were performed on a validation cohort (n = 267) where an association between large vessel size and reduced survival was also detected in ER + breast cancer (p = 0.016, log-rank test; p = 0.02; HR 2.3, 95% CI 1.1-4.7, Cox-regression analyses). CONCLUSION: Alpha-SMA/CD34 dual-IHC staining revealed breast cancer heterogeneity regarding vessel size, vessel density, and perivascular a-SMA status. Large vessel size was linked to shorter survival in ER + breast cancer.

Spatial immunophenotyping of the tumour microenvironment in non-small cell lung cancer.

INTRODUCTION: Immune cells in the tumour microenvironment are associated with prognosis and response to therapy. We aimed to comprehensively characterise the spatial immune 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+ 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.

Fibroblast subsets in non-small cell lung cancer: Associations with survival, mutations, and immune features.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are molecularly heterogeneous mesenchymal cells that interact with malignant cells and immune cells and confer anti- and protumorigenic functions. Prior in situ profiling studies of human CAFs have largely relied on scoring single markers, thus presenting a limited view of their molecular complexity. Our objective was to study the complex spatial tumor microenvironment of non-small cell lung cancer (NSCLC) with multiple CAF biomarkers, identify novel CAF subsets, and explore their associations with patient outcome. METHODS: Multiplex fluorescence immunohistochemistry was employed to spatially profile the CAF landscape in 2 population-based NSCLC cohorts (n = 636) using antibodies against 4 fibroblast markers: platelet-derived growth factor receptor-alpha (PDGFRA) and -beta (PDGFRB), fibroblast activation protein (FAP), and alpha-smooth muscle actin (αSMA). The CAF subsets were analyzed for their correlations with mutations, immune characteristics, and clinical variables as well as overall survival. RESULTS: Two CAF subsets, CAF7 (PDGFRA-/PDGFRB+/FAP+/αSMA+) and CAF13 (PDGFRA+/PDGFRB+/FAP-/αSMA+), showed statistically significant but opposite associations with tumor histology, driver mutations (tumor protein p53 [TP53] and epidermal growth factor receptor [EGFR]), immune features (programmed death-ligand 1 and CD163), and prognosis. In patients with early stage tumors (pathological tumor-node-metastasis IA-IB), CAF7 and CAF13 acted as independent prognostic factors. CONCLUSIONS: Multimarker-defined CAF subsets were identified through high-content spatial profiling. The robust associations of CAFs with driver mutations, immune features, and outcome suggest CAFs as essential factors in NSCLC progression and warrant further studies to explore their potential as biomarkers or therapeutic targets. This study also highlights multiplex fluorescence immunohistochemistry-based CAF profiling as a powerful tool for the discovery of clinically relevant CAF subsets.

Spatial genomics maps the structure, nature and evolution of cancer clones.

Genome sequencing of cancers often reveals mosaics of different subclones present in the same tumour1-3. Although these are believed to arise according to the principles of somatic evolution, the exact spatial growth patterns and underlying mechanisms remain elusive4,5. Here, to address this need, we developed a workflow that generates detailed quantitative maps of genetic subclone composition across whole-tumour sections. These provide the basis for studying clonal growth patterns, and the histological characteristics, microanatomy and microenvironmental composition of each clone. The approach rests on whole-genome sequencing, followed by highly multiplexed base-specific in situ sequencing, single-cell resolved transcriptomics and dedicated algorithms to link these layers. Applying the base-specific in situ sequencing workflow to eight tissue sections from two multifocal primary breast cancers revealed intricate subclonal growth patterns that were validated by microdissection. In a case of ductal carcinoma in situ, polyclonal neoplastic expansions occurred at the macroscopic scale but segregated within microanatomical structures. Across the stages of ductal carcinoma in situ, invasive cancer and lymph node metastasis, subclone territories are shown to exhibit distinct transcriptional and histological features and cellular microenvironments. These results provide examples of the benefits afforded by spatial genomics for deciphering the mechanisms underlying cancer evolution and microenvironmental ecology.

Tumour-infiltrating lymphocytes add prognostic information for patients with low-risk DCIS: findings from the SweDCIS randomised radiotherapy trial.

BACKGROUND: The immune microenvironment is an important modulator of tumour progression and treatment response. In invasive breast cancer, assessment of tumour-infiltrating lymphocytes (TILs) provides prognostic and predictive information. However, the clinical impact of TILs for ductal carcinoma in situ (DCIS) has not yet been demonstrated. PATIENTS AND METHODS: Post hoc analysis of the SweDCIS randomised radiotherapy trial including primary DCIS cases following breast-conserving surgery. TILs were assessed on haematoxylin-eosin sections (n = 711) according to the International Immuno-Oncology Biomarker Working Group guidelines. TILs-scores were analysed as continuous and dichotomised (≤5% versus >5%) variable regarding ipsilateral breast events (IBEs) as the predefined primary endpoint. RESULTS: Most women (61.9%) showed a TILs prevalence of ≤5%. High TILs-scores were associated with larger lesion size, human epidermal growth factor receptor 2 (HER2)-positivity, higher nuclear grade, and KI67-score. DCIS cases with high TILs prevalence had a significant increased cumulative IBE incidence at five years post-surgery (TILslow-versus TILshigh 9% versus 18%; p < 0.001). Among patients with HER2-negative DCIS, high TILs remained an independent poor prognosis marker for IBE risk in multivariable analysis with an adjusted hazard ratio of 2.41 [95%CI 1.17-4.95, p = 0.017]. Including TILs-status provided a refined stratification of patients with general low-risk DCIS (grade <3, size <25 mm, free margin). No interaction between TILs and radiotherapy benefits was detected. CONCLUSION: High TILs are associated with higher IBE risk over 5-years post-surgery, particularly for HER2-negative DCIS. Our data indicate that TILs should be integrated into the clinical workup to define patients with low-risk DCIS who can omit adjuvant therapy or patients with potential benefits from immunotherapy.

Comparison of ROS1-rearrangement detection methods in a cohort of surgically resected non-small cell lung carcinomas.

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=401, squamous cell carcinomas (SCC) n=213, other NSCLC n=62]. ROS1-rearrangements 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.

TGF-ß-mediated epithelial-mesenchymal transition and tumor-promoting effects in CMT64 cells are reflected in the transcriptomic signature of human lung adenocarcinoma.

Epithelial-mesenchymal transition (EMT) is a cellular process during which epithelial cells acquire mesenchymal phenotypes. Cancer cells undergo EMT to acquire malignant features and TGF-ß is a key regulator of EMT. Here, we demonstrate for the first time that TGF-ß could elicit EMT in a mouse lung adenocarcinoma cell line. TGF-ß signaling activation led to cell morphological changes corresponding to EMT and enhanced the expression of mesenchymal markers and EMT-associated transcription factors in CMT64 lung cancer cells. RNA-sequencing analyses revealed that TGF-ß increases expression of Tead transcription factors and an array of Tead2 target genes. TGF-ß stimulation also resulted in alternative splicing of several genes including Cd44, tight junction protein 1 (Tjp1), and Cortactin (Cttn). In parallel with EMT, TGF-ß enhanced cell growth of CMT64 cells and promoted tumor formation in a syngeneic transplantation model. Of clinical importance, the expression of TGF-ß-induced genes identified in CMT64 cells correlated with EMT gene signatures in human lung adenocarcinoma tissue samples. Furthermore, TGF-ß-induced gene enrichment was related to poor prognosis, underscoring the tumor-promoting role of TGF-ß signaling in lung adenocarcinoma. Our cellular and syngeneic transplantation model would provide a simple and useful experimental tool to study the significance of TGF-ß signaling and EMT.

Stabilization of the classical phenotype upon integration of pancreatic cancer cells into the duodenal epithelium.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid tumors. Based on transcriptomic classifiers, basal-like and classical PDAC subtypes have been defined that differ in prognosis. Cells of both subtypes can coexist in individual tumors; however, the contribution of either clonal heterogeneity or microenvironmental cues to subtype heterogeneity is unclear. Here, we report the spatial tumor phenotype dynamics in a cohort of patients in whom PDAC infiltrated the duodenal wall, and identify the duodenal epithelium as a distinct PDAC microniche. MATERIALS AND METHODS: We used serial multiplex quantitative immunohistochemistry (smq-IHC) for 24 proteins to phenotypically chart PDAC tumor cells in patients whose tumors infiltrated the duodenal epithelium. Additionally, we used a genetically engineered mouse model to study the PDAC cell phenotype in the small intestinal epithelium in a controlled genetic background. RESULT: We show that pancreatic cancer cells revert to non-destructive growth upon integration into the duodenal epithelium, where they adopt traits of intestinal cell differentiation, associated with phenotypical stabilization of the classical subtype. The integrated tumor cells replace epithelial cells in an adenoma-like manner, as opposed to invasive growth in the submucosa. Finally, we show that this phenomenon is shared between species, by confirming duodenal integration and phenotypic switching in a genetic PDAC mouse model. DISCUSSION: Our results identify the duodenal epithelium as a distinct PDAC microniche and tightly link microenvironmental cue to cancer transcriptional subtypes. The phenomenon of "intestinal mimicry" provides a unique opportunity for the systematic investigation of microenvironmental influences on pancreatic cancer plasticity.

Infiltration of NK and plasma cells is associated with a distinct immune subset in non-small cell lung cancer.

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.

Multiplexed Imaging for Immune Profiling on Human FFPE Material.

In this chapter, we describe the pipeline for multiplex immunohistochemical staining, multispectral image acquisition, and analysis. The protocol is dedicated for use on human formalin fixed paraffin embedded (FFPE) tissues and utilizes immune markers of dendritic cells, myeloid cells, and macrophages, as well as cytokeratin. This provides quantitative data of the (co-)expression levels and spatial localization of immune cell subtypes.

High PDGFRb Expression Predicts Resistance to Radiotherapy in DCIS within the SweDCIS Randomized Trial.

PURPOSE: This study analyzes the potential of stromal platelet-derived growth factor receptor-beta (PDGFRb) expression as biomarker for radiotherapy (RT) benefit on ipsilateral breast events (IBE) in ductal carcinoma in situ (DCIS). Improved identification of DCIS patients refractory to adjuvant whole-breast RT is needed. Predictive biomarker studies in DCIS have focused on tumor cell features rather than the tumor-associated stroma, despite growing evidence of its influence on therapy efficiency. EXPERIMENTAL DESIGN: Samples from the Swedish randomized radiotherapy DCIS trial (SweDCIS) were subjected to IHC analysis for stromal PDGFRb expression. IBE incidence at 10 years after breast-conserving surgery was the primary endpoint. Interactions between marker and treatment were analyzed. RESULTS: PDGFRb score was predictive for RT benefit with regard to IBE (P interaction = 0.002 and P interaction = 0.008 adjusted multivariably). Patients of the PDGFRblow group had a strong benefit from RT regarding IBE risk [HR, 0.23; 95% confidence interval (CI), 0.12-0.45; P < 0.001] with an absolute risk reduction of 21% (cumulative risk 7% vs. 28%) at 10 years. No significant risk reduction by RT was observed for patients of the PDGFRbhigh group (HR, 0.83; 0.51-1.34; P = 0.444; cumulative risk 22% vs. 25%). The RT response-predictive effect of stromal PDGFRb was equally strong in analyses for in situ and invasive IBE when analyzed separately (in situ IBE: P = 0.029; invasive IBE: P = 0.044). CONCLUSIONS: Results suggest high stromal PDGFRb expression as a novel biomarker identifying DCIS patients who are refractory to standard whole-breast adjuvant RT. The data imply previously unrecognized fibroblast-mediated modulation of radiosensitivity of DCIS, which should be further explored from mechanistic and targeting perspectives.

FGFR1 overexpression in non-small cell lung cancer is mediated by genetic and epigenetic mechanisms and is a determinant of FGFR1 inhibitor response.

Amplification of fibroblast growth factor receptor 1 (FGFR1) in non-small cell lung cancer (NSCLC) has been considered as an actionable drug target. However, pan-FGFR tyrosine kinase inhibitors did not demonstrate convincing clinical efficacy in FGFR1-amplified NSCLC patients. This study aimed to characterise the molecular context of FGFR1 expression and to define biomarkers predictive of FGFR1 inhibitor response. In this study, 635 NSCLC samples were characterised for FGFR1 protein expression by immunohistochemistry and copy number gain (CNG) by in situ hybridisation (n = 298) or DNA microarray (n = 189). FGFR1 gene expression (n = 369) and immune cell profiles (n = 309) were also examined. Furthermore, gene expression, methylation and microRNA data from The Cancer Genome Atlas (TCGA) were compared. A panel of FGFR1-amplified NSCLC patient-derived xenograft (PDX) models were tested for response to the selective FGFR1 antagonist M6123. A minority of patients demonstrated FGFR1 CNG (10.5%) or increased FGFR1 mRNA (8.7%) and protein expression (4.4%). FGFR1 CNG correlated weakly with FGFR1 gene and protein expression. Tumours overexpressing FGFR1 protein were typically devoid of driver alterations (e.g. EGFR, KRAS) and showed reduced infiltration of T-lymphocytes and lower PD-L1 expression. Promoter methylation and microRNA were identified as regulators of FGFR1 expression in NSCLC and other cancers. Finally, NSCLC PDX models demonstrating FGFR1 amplification and FGFR1 protein overexpression were sensitive to M6123. The unique molecular and immune features of tumours with high FGFR1 expression provide a rationale to stratify patients in future clinical trials of FGFR1 pathway-targeting agents.

Stromal FAP is an independent poor prognosis marker in non-small cell lung adenocarcinoma and associated with p53 mutation.

OBJECTIVES: Fibroblasts regulate tumor growth and immune surveillance. Here, we study FAP, PDGFßR and α-SMA fibroblast markers in a well-annotated clinical cohort of non-small-cell lung cancer (NSCLC) for analyses of associations with immune cell infiltration, mutation status and survival. MATERIALS AND METHODS: A well-annotated NSCLC cohort was subjected to IHC analyses of stromal expression of FAP, PDGFßR and α-SMA and of stromal CD8 density. Fibroblast markers-related measurements were analyzed with regard to potential associations with CD8 density, cancer genetic driver mutations, survival and PD-L1 expression in the whole NSCLC cohort and in subsets of patients. RESULTS: High stromal FAP expression was identified as an independent poor prognostic marker in the whole study population (HR 1.481; 95 % CI, 1.012-2.167, p = 0.023) and in the histological subset of adenocarcinoma (HR 1.720; 95 % CI, 1.126-2.627, p = 0.012). Among patients with adenocarcinoma, a particularly strong association of FAP with poor survival was detected in patients with low stromal CD8 infiltration, and in other subpopulations identified by specific clinical characteristics; elderly patients, females, non-smokers and patients with normal ECOG performance status. α-SMA expression was negatively associated with CD8 infiltration in non-smokers, but none of the fibroblast markers expression was associated with CD8 density in the whole study population. Significant associations were detected between presence of p53 mutations and high α-SMA (p = 0.003) and FAP expression (p < 0.001). CONCLUSION: The study identifies FAP intensity as a candidate independent NSCLC prognostic biomarker. The study also suggests continued analyses of the relationships between genetic driver mutations and the composition of tumor stroma, as well as continued probing of marker-defined fibroblasts as NSCLC subset-specific modifiers of immune surveillance and outcome.

The prognostic impact of the tumour stroma fraction: A machine learning-based analysis in 16 human solid tumour types.

BACKGROUND: The development of a reactive tumour stroma is a hallmark of tumour progression and pronounced tumour stroma is generally considered to be associated with clinical aggressiveness. The variability between tumour types regarding stroma fraction, and its prognosis associations, have not been systematically analysed. METHODS: Using an objective machine-learning method we quantified the tumour stroma in 16 solid cancer types from 2732 patients, representing retrospective tissue collections of surgically resected primary tumours. Image analysis performed tissue segmentation into stromal and epithelial compartment based on pan-cytokeratin staining and autofluorescence patterns. FINDINGS: The stroma fraction was highly variable within and across the tumour types, with kidney cancer showing the lowest and pancreato-biliary type periampullary cancer showing the highest stroma proportion (median 19% and 73% respectively). Adjusted Cox regression models revealed both positive (pancreato-biliary type periampullary cancer and oestrogen negative breast cancer, HR(95%CI)=0.56(0.34-0.92) and HR(95%CI)=0.41(0.17-0.98) respectively) and negative (intestinal type periampullary cancer, HR(95%CI)=3.59(1.49-8.62)) associations of the tumour stroma fraction with survival. INTERPRETATION: Our study provides an objective quantification of the tumour stroma fraction across major types of solid cancer. Findings strongly argue against the commonly promoted view of a general associations between high stroma abundance and poor prognosis. The results also suggest that full exploitation of the prognostic potential of tumour stroma requires analyses that go beyond determination of stroma abundance. FUNDING: The Swedish Cancer Society, The Lions Cancer Foundation Uppsala, The Swedish Government Grant for Clinical Research, The Mrs Berta Kamprad Foundation, Sweden, Sellanders foundation, P.O.Zetterling Foundation, and The Sjöberg Foundation, Sweden.

Prognostic and predictive impact of stroma cells defined by PDGFRb expression in early breast cancer: results from the randomized SweBCG91RT trial.

PURPOSE: Predictive biomarkers are needed to aid the individualization of radiotherapy (RT) in breast cancer. Cancer-associated fibroblasts have been implicated in tumor radioresistance and can be identified by platelet-derived growth factor receptor-beta (PDGFRb). This study aims to analyze how PDGFRb expression affects RT benefit in a large randomized RT trial. METHODS: PDGFRb was assessed by immunohistochemistry on tissue microarrays from 989 tumors of the SweBCG91RT trial, which enrolled lymph node-negative, stage I/IIA breast cancer patients randomized to RT after breast-conserving surgery. Outcomes were analyzed at 10 years for ipsilateral breast tumor recurrence (IBTR) and any recurrence and 15 years for breast cancer specific death (BCSD). RESULTS: PDGFRb expression correlated with estrogen receptor negativity and younger age. An increased risk for any recurrence was noted in univariable analysis for the medium (HR 1.58, CI 95% 1.11-2.23, p = 0.011) or PDGFRb high group (1.49, 1.06-2.10, p = 0.021) compared to the low group. No differences in IBTR or BCSD risk were detected. RT benefit regarding IBTR risk was significant in the PDGFRb low (0.29, 0.12-0.67, p = 0.004) and medium (0.31, 0.16-0.59, p < 0.001) groups but not the PDGFRb high group (0.64, 0.36-1.11, p = 0.110) in multivariable analysis. Likewise, risk reduction for any recurrence was less pronounced in the PDGFRb high group. No significant interaction between RT and PDGFRb-score could be detected. CONCLUSION: A higher PDGFRb-score conferred an increased risk of any recurrence, which partly can be explained by its association with estrogen receptor negativity and young age. Reduced RT benefit was noted among patients with high PDGFRb, however without significant interaction.

PD-L1 amplification is associated with an immune cell rich phenotype in squamous cell cancer of the lung.

Gene amplification is considered to be one responsible cause for upregulation of Programmed Death Ligand-1 (PD-L1) in non-small cell lung cancer (NSCLC) and to represent a specific molecular subgroup possibly associated with immunotherapy response. Our aim was to analyze the frequency of PD-L1 amplification, its relation to PD-L1 mRNA and protein expression, and to characterize the immune microenvironment of amplified cases. The study was based on two independent NSCLC cohorts, including 354 and 349 cases, respectively. Tissue microarrays were used to evaluate PD-L1 amplification by FISH and PD-L1 protein by immunohistochemistry. Immune infiltrates were characterized immunohistochemically by a panel of immune markers (CD3, CD4, CD8, PD-1, Foxp3, CD20, CD138, CD168, CD45RO, NKp46). Mutational status was determined by targeted sequencing. RNAseq data was available for 197 patients. PD-L1 amplification was detected in 4.5% of all evaluable cases. PD-L1 amplification correlated only weakly with mRNA and protein expression. About  37% of amplified cases were negative for PD-L1 protein. PD-L1 amplification did not show any association with the mutational status. In squamous cell cancer, PD-L1 amplified cases were enriched among patients with high tumoral immune cell infiltration and showed gene expression profiles related to immune exhaustion. In conclusion, PD-L1 amplification correlates with PD-L1 expression in squamous cell cancer and was associated with an immune cell rich tumor phenotype. The correlative findings help to understand the role of PD-L1 amplification as an important immune escape mechanism in NSCLC and suggest the need to further evaluate PD-L1 amplification as predictive biomarker for checkpoint inhibitor therapy.

Evaluation of NTRK immunohistochemistry as a screening method for NTRK gene fusion detection in non-small cell lung cancer.

PURPOSE: The small molecule inhibitors larotrectinib and entrectinib have recently been approved as cancer agnostic drugs in patients with tumours harbouring a rearrangement of the neurotrophic tropomyosin receptor kinase (NTRK). These oncogenic fusions are estimated to occur in 0.1-3 % of non-small cell lung cancers (NSCLC). Although molecular techniques are most reliable for fusion detection, immunohistochemical analysis is considered valuable for screening. Therefore, we evaluated the newly introduced diagnostic immunohistochemical assay (clone EPR17341) on a representative NSCLC cohort. METHODS: Cancer tissue from 688 clinically and molecularly extensively annotated NSCLC patients were comprised on tissue microarrays and stained with the pan-TRK antibody clone EPR17341. Positive cases were further analysed with the TruSight Tumor 170 RNA assay (Illumina). Selected cases were also tested with a NanoString NTRK fusion assay. For 199 cases, NTRK RNA expression data were available from previous RNA sequencing analysis. RESULTS: Altogether, staining patterns for 617 NSCLC cases were evaluable. Of these, four cases (0.6 %) demonstrated a strong diffuse cytoplasmic and membranous staining, and seven cases a moderate staining (1.1 %). NanoString or TST170-analysis could not confirm an NTRK fusion in any of the IHC positive cases, or any of the cases with high mRNA levels. In the four cases with strong staining intensity in the tissue microarray, whole section staining revealed marked heterogeneity of NTRK protein expression. CONCLUSION: The presence of NTRK fusion genes in non-small cell lung cancer is exceedingly rare. The use of the immunohistochemical NTRK assay will result in a small number of false positive cases. This should be considered when the assay is applied as a screening tool in clinical diagnostics.