Thomas Karsten Kilvær

Stromal CD8+ T-cell Density—A Promising Supplement to TNM Staging in Non–Small Cell Lung Cancer

Purpose: Immunoscore is a prognostic tool defined to quantify in situ immune cell infiltrates, which appears to be superior to the tumor–node–metastasis (TNM) classification in colorectal cancer. In non–small cell lung cancer (NSCLC), no immunoscore has been established, but in situ tumor immunology is recognized as highly important. We have previously evaluated the prognostic impact of several immunological markers in NSCLC, yielding the density of stromal CD8+ tumor-infiltrating lymphocytes (TIL) as the most promising candidate. Hence, we validate the impact of stromal CD8+ TIL density as an immunoscore in NSCLC. Experimental Design: The prognostic impact of stromal CD8+ TILs was evaluated in four different cohorts from Norway and Denmark consisting of 797 stage I–IIIA NSCLC patients. The Tromso cohort (n = 155) was used as training set, and the results were further validated in the cohorts from Bodo (n = 169), Oslo (n = 295), and Denmark (n = 178). Tissue microarrays and clinical routine CD8 staining were used for all cohorts. Results: Stromal CD8+ TIL density was an independent prognostic factor in the total material (n = 797) regardless of the endpoint: disease-free survival (P < 0.001), disease-specific survival (P < 0.001), or overall survival (P < 0.001). Subgroup analyses revealed significant prognostic impact of stromal CD8+ TIL density within each pathologic stage (pStage). In multivariate analysis, stromal CD8+ TIL density and pStage were independent prognostic variables. Conclusions: Stromal CD8+ TIL density has independent prognostic impact in resected NSCLC, adds prognostic impact within each pStage, and is a good candidate marker for establishing a TNM-Immunoscore. Clin Cancer Res; 21(11); 2635–43. ©2015 AACR.

Strategies for clinical implementation of TNM-Immunoscore in resected nonsmall-cell lung cancer.

Immunoscore is a prognostic tool defined to quantify in situ immune cell infiltrates and appears highly promising as a supplement to the tumor-node-metastasis (TNM) classification of various tumors. In colorectal cancer, an international task force has initiated prospective multicenter studies aiming to implement TNM-Immunoscore (TNM-I) in a routine clinical setting. In breast cancer, recommendations for the evaluation of tumor-infiltrating lymphocytes (TILs) have been proposed by an international working group. Regardless of promising results, there are potential obstacles related to implementing TNM-I into the clinic. Diverse methods may be needed for different malignancies and even within each cancer entity. Nevertheless, a uniform approach across malignancies would be advantageous. In nonsmall-cell lung cancer (NSCLC), there are several previous reports indicating an apparent prognostic importance of TILs, but studies on TILs in a TNM-I setting are sparse and no general recommendations are made. However, recently published data is promising, evoking a realistic hope of a clinical useful NSCLC TNM-I. This review will focus on the TNM-I potential in NSCLC and propose strategies for clinical implementation of a TNM-I in resected NSCLC.

The Prognostic Impact of TGF-β1, Fascin, NF-κB and PKC-ζ Expression in Soft Tissue Sarcomas

Aims Transforming growth factor-β (TGF-β), fascin, nuclear factor-kappa B (NF-κB) p105, protein-kinase C-zeta (PKC-ζ), partioning-defective protein-6 (Par-6), E-cadherin and vimentin are tumor promoting molecules through mechanisms involved in cell dedifferentiation. In soft tissue sarcomas, their expression profile is poorly defined and their significance is uncertain. We aimed to investigate the prognostic impact of TGF-β1, NF-κB p105, PKC-ζ, Par-6α, E-cadherin and vimentin in non-gastrointestinal stromal tumor soft tissue sarcomas (non-GIST STSs). Patients and Methods Tumor samples and clinical data from 249 patients with non-GIST STS were obtained, and tissue microarrays (TMAs) were constructed for each specimen. Immunohistochemistry (IHC) was used to evaluate marker expression in tumor cells. Results In univariate analysis, the expression levels of TGF-β1 (P = 0.016), fascin (P = 0.006), NF-κB p105 (P = 0.022) and PKC-ζ, (P = 0.042) were significant indicators for disease specific survival (DSS). In the multivariate analysis, high TGF-β1 expression was an independent negative prognostic factor for DSS (HR = 1.6, 95% CI = 1.1–2.4, P = 0.019) in addition to tumor depth, malignancy grade, metastasis at diagnosis, surgery and positive resection margins. Conclusion Expression of TGF-β1 was significantly associated with aggressive behavior and shorter DSS in non-GIST STSs.

Profiling of VEGFs and VEGFRs as prognostic factors in soft tissue sarcoma: VEGFR-3 is an independent predictor of poor prognosis.

Background In non-gastrointestinal stromal tumor soft tissue sarcoma (non-GIST STS) optimal treatment is surgery with wide resection margins. Vascular endothelial growth factors (VEGFs) and receptors (VEGFRs) are known to be key players in the initiation of angiogenesis and lymphangiogenesis. This study investigates the prognostic impact of VEGFs and VEGFRs in non-GIST STS with wide and non-wide resection margins. Methods Tumor samples from 249 patients with non-GIST STS were obtained and tissue microarrays were constructed for each specimen. Immunohistochemistry was used to evaluate the expressions of VEGF-A, -C and -D and VEGFR-1, -2 and -3. Results In the univariate analyses, VEGF-A (P = 0.040) in the total material, and VEGF-A (P = 0.018), VEGF-C (P = 0.025) and VEGFR-3 (P = 0.027) in the subgroup with wide resection margins, were significant negative prognostic indicators of disease-specific survival (DSS). In the multivariate analysis, high expression of VEGFR-3 (P = 0.042, HR = 1.907, 95% CI 1.024-3.549) was an independent significant negative prognostic marker for DSS among patients with wide resection margins. Conclusion VEGFR-3 is a strong and independent negative prognostic marker for non-GIST STSs with wide resection margins.

Assessing PDL-1 and PD-1 in Non–Small Cell Lung Cancer: A Novel Immunoscore Approach

Introduction Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD‐1) or its ligand, PD‐L1, have gained momentum in the treatment of non–small cell lung cancer (NSCLC). However, their prognostic significance remains controversial. The present study evaluated the expression of PD‐L1 and PD‐1 and their potential role in an Immunoscore, supplementing the TNM classification of NSCLC. Materials and Methods Tissue microarrays constructed from tumor tissue samples from 2 cohorts of a total of 536 patients (University Hospital of North Norway, n = 285; Nordland Hospital, n = 251) with primary resected stage I to IIIA NSCLC. PD‐L1 and PD‐1 were evaluated by immunohistochemistry in the primary tumor and metastatic lymph node tissue. Results In univariate analysis, a high density of PD‐L1+ immune cells in the stromal compartment (S‐PD‐L1) and PD‐1+ intraepithelial tumor infiltrating lymphocytes (T‐PD‐1) was associated with favorable disease‐specific survival (DSS; S‐PD‐L1, P = .004; T‐PD‐1, P = .012), both limited to the squamous cell carcinoma histologic subgroup (S‐PD‐L1, P = .002; T‐PD‐1, P = .034). A combined low S‐PD‐L1 and T‐PD‐1 was associated with poor survival in all patients (DSS: hazard ratio [HR], 1.81; 95% confidence interval [CI], 1.37‐2.40; P < .001) at both centers and for all pathologic stages. In multivariate analysis, S‐PD‐L1 and T‐PD‐1 were independent positive prognostic factors, and combined low scores remained an independent prognosticator for poor survival (DSS: HR, 1.72; 95% CI, 1.29‐2.28; P < .001; disease‐free survival, P = .001; overall survival, P = .005). Conclusion Our study identified S‐PD‐L1 and T‐PD‐1 as independent positive prognostic factors for NSCLC patients. Their combination added significant prognostic impact within each pathologic stage and hence are feasible to include in a TNM Immunoscore. Micro‐Abstract Novel immune biomarkers could complement the TNM classification for non–small cell cancer (NSCLC), improving the prognostic accuracy. The present study evaluated the prognostic significance of the immune checkpoint molecules programmed cell death protein 1 (PD‐1) and PD‐1 ligand (PD‐L1) in 536 patients with stage I to IIIA NSCLC using an Immunoscore approach. Independently, and in combination, the infiltration of immune cells expressing PD‐L1 and PD‐1 predicted patient survival, supplementing the TNM classification in each stage.

Cancer associated fibroblasts in stage I-IIIA NSCLC: Prognostic impact and their correlations with tumor molecular markers

Background Cancer Associated Fibroblasts (CAFs) are thought to regulate tumor growth and metastasis. Fibroblast Activating Protein 1 (FAP-1) is a marker for fibroblast activation and by many recognized as the main marker of CAFs. Alpha Smooth Muscle Actin (α-SMA) is a general myofibroblast marker, and can be used to identify CAFs. This study investigates the prognostic impact of FAP-1 and α-SMA in non-small cell lung cancer (NSCLC) patients and correlates their expression to 105 proteins investigated in the same cohort. Methods Tumor specimens from 536 NSCLC patients were obtained and tissue micro-arrays were constructed. Immunohistochemistry was used to evaluate the expression of FAP-1 and α-SMA and explore their impact on survival and association with other tumor molecular markers in NSCLC patients. Results High expression of FAP-1, but not α-SMA, in squamous cell carcinoma (SCC, P = 0.043, HR = 0.63 95% CI 0.40–0.99) was significantly associated with increased disease-specific survival. FAP-1 and α-SMA were not significantly correlated to each other. Analyses of FAP-1 and α-SMA associated with other tumor-related proteins revealed histotype-specific correlation patterns. Conclusion The presence of FAP-1 expressing CAFs is an indicator of positive outcome for NSCLC-SCC patients. In addition, correlation analyses suggest FAP-1 and α-SMA to label different subsets of fibroblasts and their associations with other tumor-related proteins diverge according to histological subtype.

CD45RO+ Memory T Lymphocytes — a Candidate Marker for TNM-Immunoscore in Squamous Non–Small Cell Lung Cancer

Tumor-infiltrating lymphocytes (TILs) are vital in limiting cancer progression and may supplement the TNM classification. CD45RO+ memory TILs show major prognostic impact in various malignancies but have not been extensively explored in non–small cell lung cancer (NSCLC). In this study, we aimed to evaluate their potential in a NSCLC TNM-Immunoscore. Tissue microarrays were constructed from tumor tissue samples from two cohorts including in total 536 patients (University Hospital of North Norway, n = 285; Nordland Hospital, n = 251) with primary resected stage I to IIIA NSCLC. The density of CD45RO+ and CD8+ TILs in tumor epithelial and stromal compartments of the tumors was evaluated by immunohistochemistry. In univariate analyses, intraepithelial CD45RO+ TIL density (T-CD45RO) was a significant prognostic factor for disease-specific survival (P = .007), limited to the squamous cell carcinoma (SCC) histology subgroup (P < .001), where it was significant in both cohorts (University Hospital of North Norway, P = .003; Nordland Hospital, P = .022). Combining T-CD45RO and stromal CD8+ TIL density (S-CD8) increased the prognostic impact in SCC (P < .001) and showed a significant impact within all pathological stages (I, P = .025; II, P < .001; III, P = .001). In the multivariate analysis, T-CD45RO was an independent positive prognostic factor for SCC (hazard ratio 2.65, 95% confidence interval 1.64-4.28, P < .001), and in combination with S-CD8, the prognostic impact increased vastly (high + high versus low + low: hazard ratio 6.50, 95% confidence interval 3.54-11.91, P < .001). In conclusion, T-CD45RO was an independent prognostic factor for SCC NSCLC. When combined with S-CD8, the prognostic impact increased and was significant within each pathological stage. We propose CD45RO as a candidate marker for TNM-Immunoscore in SCC NSCLC.

The prognostic impact of Akt isoforms, PI3K and PTEN related to female steroid hormone receptors in soft tissue sarcomas

BackgroundThe PI3K/Akt pathway is involved in cellular survival pathways by inhibiting apoptotic processes and stimulating cell growth and proliferation. Its negative prognostic value has been proven in many types of cancer. In soft tissue sarcomas, the expression profiles of the PI3K/Akt pathway components are poorly defined and their significance uncertain. We aimed to investigate the prognostic impact of Akt (Akt1) phosphorylated at threonine308 and serine473, Akt2, Akt3, PI3K and PTEN, alone and in coexpression with ER and PgR in non-gastrointestinal stromal tumor soft tissue sarcomas (non-GIST STSs).Patients and methodsTumor samples and clinical data from 249 patients with non-GIST STS were obtained, and tissue microarrays (TMAs) were constructed. Immunohistochemistry (IHC) was used to evaluate marker expression in tumor cells.ResultsIn univariate analyses, the expression levels of p-Akt Thr308 (P = 0.002), Akt2 (P = 0.008) and PI3K (P < 0.001) were significant prognostic factors. In the multivariate analysis, high PI3K expression was an independent negative prognosticator (HR = 1.5, 95% CI = 1.0-2.2, P = 0.042) in addition to advanced age, tumor depth, high malignancy grade, metastasis at diagnosis, surgery and positive resection margins. p-Akt Thr308 expression had strong unfavorable effect in men only (P = 0.009). In contrast, p-Akt Ser473 expression had strong unfavorable impact in women (P = 0.023). PgR-/p-Akt Ser473+ phenotype tended to have less favorable impact in women (P = 0.087), but was the most favorable one in men (P = 0.010).ConclusionExpression of PI3K was significantly associated with aggressive behavior and shorter DSS in non-GIST STSs. The site of Akt phosphorylation seems to have gender-dependent impact on survival in STS patients.

Fibroblast growth factor 2 orchestrates angiogenic networking in non-GIST STS patients

BackgroundNon-gastrointestinal stromal tumor soft-tissue sarcomas (non-GIST STSs) constitute a heterogeneous group of tumors with poor prognosis. Fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor-1 (FGFR-1), in close interplay with platelet-derived growth factor-B (PDGF-B) and vascular endothelial growth factor receptor-3 (VEGFR-3), are strongly involved in angiogenesis. This study investigates the prognostic impact of FGF2 and FGFR-1 and explores the impact of their co-expression with PDGF-B and VEGFR-3 in widely resected tumors from non-GIST STS patients.MethodsTumor samples from 108 non-GIST STS patients were obtained and tissue microarrays were constructed for each specimen. Immunohistochemistry was used to evaluate the expressions of FGF-2, FGFR-1, PDGF-B and VEGFR-3.ResultsIn the multivariate analysis, high expression of FGF2 (P = 0.024, HR = 2.2, 95% CI 1.1-4.4) and the co-expressions of FGF2 & PDGF-B (overall; P = 0.007, intermediate; P = 0.013, HR = 3.6, 95% CI = 1.3-9.7, high; P = 0.002, HR = 6.0, 95% CI = 2.0-18.1) and FGF2 & VEGFR-3 (overall; P = 0.050, intermediate; P = 0.058, HR = 2.0, 95% CI = 0.98-4.1, high; P = 0.028, HR = 2.6, 95% CI = 1.1-6.0) were significant independent prognostic indicators of poor disease-specific survival.ConclusionFGF2, alone or in co-expression with PDGF-B and VEGFR-3, is a significant independent negative prognosticator in widely resected non-GIST STS patients.

Prognostic impact of peritumoral lymphocyte infiltration in soft tissue sarcomas

BackgroundThe purpose of this study was to clarify the prognostic significance of peritumoral lymphocyte infiltration in the capsule of soft tissue sarcomas (STS). Multiple observations in preclinical and clinical studies have shown that the immune system has a role in controlling tumor growth and progression. Prognostic markers in potentially curable STS should guide therapy after surgical resection. The immune status at the time of resection may be important, but the prognostic significance of peritumoral lymphocytes is unknown.MethodsTissue microarrays from 80 patients with STS were constructed from duplicate cores of tissue from the tumor and the peritumoral capsule. Immunohistochemistry was used to evaluate the CD3+, CD4+, CD8+ and CD20+ lymphocytes in the tumor and the peritumoral capsule.ResultsIn univariate analyses, increasing numbers of CD20+ (P = 0.032) peritumoral lymphocytes were associated with a reduced disease free survival (DSS). In multivariate analyses, a high number of CD20+ peritumoral lymphocytes (P = 0.030) in the capsule was an independent negative prognostic factor for DSS. There were no such associations of lymphocyte infiltration in the tumor.ConclusionsA high density of CD20+ peritumoral lymphocytes is an independent negative prognostic indicator for patients with STS. Further research is needed to determine whether CD20 cells in the peritumoral capsule of STS may promote tumor invasion in the surrounding tissue and increase the metastatic potential.