Tumor Invasive Margin Research Articles

Tumor budding in laryngeal carcinoma.

Tumor budding was defined as a single cancer cell or a cluster of fewer than five cancer cells in the stroma of the invasive tumor margin. It has been suggested as a prognostic factor in various cancers, such as esophageal, lung, colorectal, and endometrial. There are only a few studies about the prognostic signifi cance of tumor budding in laryngeal carcinomas. A total of 81 patients with laryngeal carcinoma diagnosed between 2011 and 2016 and treated by partial or total laryngectomy were evaluated. Clinicopathologic parameters were correlated with the presence and grade of tumor budding. The study was consisted of 77 (95.1%) male and 4 (4.9%) female patients. The mean age of the patients was 60.2 years (min: 42 and max: 78). Median follow-up time was 25 months (min: 7 and max: 54) (SD ±11.5). Histopathologic diagnosis was squamous cell carcinoma (SCC) in all patients. Of the 62 cases showing budding, 2 (3.2%) were stage 1, 12 (19.4%) stage 2, 16 (25.8%) stage 3, and 32 (51.6%) were stage 4. Fifteen cases with budding (24.2%) showed lymphovascular invasion (LVI). None of the nonbudding cases had LVI and perineural invasion (PNI). Statistical analysis revealed that LVI and PNI were signifi cantly associated with budding (P = 0.017 and P = 0.012). Among the tumors showing budding, 37% had lymph node metastasis (LNM). In nonbudding cases 15% had LNM. There was a statistically significant correlation between LNM and budding (P = 0.017). None of the parameters correlated with grade of tumor budding statistically. The results of this study suggest that tumor budding might be used as a prognostic factor in laryngeal SCCs.

Presence of lymphocytic infiltrate cytotoxic T lymphocyte CD3+, CD8+, and immunoscore as prognostic marker in patients after radical cystectomy.

Tumor-Infiltrating Lymphocytes (TILs) has been shown to be essential to predict disease outcome in several types of cancers. Moreover, the distribution of cytotoxic T lymphocytes (CD8+) and T cells in general (CD3+) have been used to establish an Immunoscore, as a new cancer prognosticator for survival in colon and lung cancer. In bladder cancer, immune activation has been shown to be associated with genomic subtypes of muscle invasive bladder cancer (MIBC). We sought to evaluate the prognostic impact of these immune cell types in MIBC patients treated with radical cystectomy. For this purpose, cystectomy sections (n = 67) with identifiable invasive margin were selected and stained for CD8+ and CD3+ tumour infiltrating lymphocytes (TILs); both tumor core (CT) and invasive margin (IM) were assessed. Immunoscore was calculated based on previously defined criteria and used to illustrate differences in survival. High density of CD8IM TILs was associated with better disease-free (DFS) (P = 0.01) and overall survival (OS) (P = 0.02) whereas CD3IM TILs were associated with better OS (P = 0.05). Immunoscore was associated with improved DFS (P = 0.02) and OS (P = 0.05). The expression of cytotoxic T cells (CD8+ T cells) in TCGA bladder cancer was also investigated from RNA-Seq profiles of 344 cases. T cell cytotoxicity associated genes (n = 113) were derived from MSig GSEA database. Luminal (n = 121) and basal (n = 68) samples were used to evaluate expression differences. Differential expression (P<0.05) of cytotoxic T cell genes was noted across different molecular subsets of bladder cancer within TCGA analysis. Our data suggests host immune system appears to play a valuable prognostic role in MIBC.

High Density of Intratumor CD45RO+ Memory Tumor-Infiltrating Lymphocytes Predicts Favorable Prognosis in Patients With Oral Squamous Cell Carcinoma

Although tumor-infiltrating lymphocytes (TILs) have been increasingly appreciated as novel biomarkers for prognostic prediction in cancer, little attention has been paid to the CD45RO+ memory TIL and its associations with clinical outcomes in oral squamous cell carcinoma (OSCC). The purpose of this study was to determine the associations between CD45RO+ TILs and clinicopathologic parameters and prognosis in OSCC. Tissue sections of primary OSCC from 2 independent tertiary referral cancer centers (Nanjing and Wuxi, China) were retrospectively collected and subjected to immunohistochemical staining for CD45RO. Densities of CD45RO+ TILs in the tumor center (CT) and invasive margin were calculated. Optimal cutoff values of CD45RO+ TILs for patient stratification were generated by X-tile software. Kaplan-Meier and Cox regression analyses were performed to assess associations between CD45RO+ TILs and overall survival and recurrence-free survival. Prognostic prediction of CD45RO+ TILs was estimated by receiver operating characteristic (ROC) curve. One hundred sixty-nine eligible patients with OSCC were included. No relevant associations between CD45RO+ TILs and clinicopathologic parameters were identified. Kaplan-Meier analyses indicated that a high density of CD45RO TILs in the CT was significantly associated with favorable overall and recurrence-free survival (P=.0018 and .0007 by log-rank test). Cox proportional regression analyses showed that presence of CD45RO TILs in the CT was an independent prognostic factor for overall survival of OSCC. ROC curves showed that presence of CD45RO TILs in the CT was comparable to clinical stage in predicting patient survival, whereas their combination was superior to either parameter alone. The present findings indicate that intratumor density of CD45RO TILs is a viable and independent prognostic predictor for OSCC.

SERPINE1 IDENTIFIED AS POTENTIAL THERAPEUTIC TARGET THROUGH RNA-SEQ OF CLINICALLY RELEVANT INVASIVE GLIOBLASTOMA CELLS ISOLATED FROM PATIENTS BY 5-ALA BASED METHODOLOGY

We previously described a method to separate residual tumour cells from normal brain using fluorescent activated cell sorting (FACS). Using RNA-Seq on these cells we have identified new potential targets including SERPINE1, whose expression remains high in tumour cells as they invade into normal tissue. 5-aminolevulinic acid (5-ALA) is a clinically used drug that facilitates fluorescence guided resection of glioblastoma (GBM). 11 tumour samples were dissociated and FACS used to separate residual fluorescent cancer cells (1%) from within normal brain. FACS sorted and unsorted mixed samples from tumour core, rim and invasive margin were compared. Gene expression was analysed by RNA-Seq and validated by qPCR, IHC and in vivo xenografts. Differential expression analysis identified 2567 genes with differences between core and invasive margin, and 78 genes with differences between 5-ALA FACS positive and FACS negative. Interestingly SERPINE1 expression is reduced in the unsorted invasive margin but expression remains high within sorted invasive tumour cells. Pathway analysis identified a predominance of immune system pathway changes between core and invasive margin. The differential expression of SERPINE1, VEGF, CHI3L1 and RTN1 in qPCR and IHC validated the same changes observed from the RNA-Seq data. The invasive and tumorigenic capacity of 5-ALA positive sorted tumour cells was confirmed by enhanced engraftment in a mouse flank model compared to unsorted cells, whereas non 5-ALA sorted cells failed to engraft. This study has demonstrated that 5-ALA fluorescent sorting of tumour cells from the invasive margin can identify new targets such as SERPINE1, whose high expression in invasive tumour cells would otherwise be overlooked. Our approach gives hope that we can interrogate the true residual disease, and for the first time gain insight into the source of tumour recurrence.

P04.09 MicroRNA analysis of the invasive margin of Glioblastoma reveals druggable therapeutic targets in lipid metabolism pathways

Glioblastoma (GBM) is the most common type of adult brain tumour, representing 45.2% of all malignant brain and central nervous system tumours. It is also the most lethal, with a median post-therapy survival of 14 months, due to the high likelihood of tumour recurrence. Intratumour heterogeneity is thought to be a key contributor to GBM tumour recurrence, because treatments such as radiotherapy and chemotherapy typically only successfully target the core of the tumour. Due to its high visibility, this core is excised during surgery, but there is another more subtle region of the tumour, the invasive margin, which generally remains behind. This invasive margin is a mixture between normal brain and highly invasive tumour cells. If left over from surgery, these remaining tumour cells infiltrate the normal brain, and cause recurrence within 2cm of the original site. However, if surgeons are too vigorous in removing the invasive margin, they may cause unnecessary removal of healthy brain tissue, thereby causing other complications. This make developing specific methods to target the cancer cells in the invasive margin a priority area in GBM research. In order to specifically target the invasive margin, we need to have a way of differentiating it reliably from the other regions. In this thesis, I investigated the role that micro RNAs (miRNAs), a type of small non-coding RNA, play in this intratumour heterogeneity. miRNAs act as post-transcriptional regulators of gene expression and can simultaneously suppress multiple genes within the same pathway, which makes them potentially valuable routes for therapy development. I performed a miRNA microarray on tissue samples from three tumour regions (tumour core, rim, and invasive margin) and validated the results with qRT-PCR. I then performed computational predictions of gene targets of the differentially expressed miRNAs and validated them in cell lines and patient samples at both the mRNA (qRT-PCR) and protein (Western blot and immunohistochemistry) levels. I then performed a bi-phasic extraction of polar and non-polar metabolites and employed liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectroscopy techniques to profile the intracellular metabolome and lipidome of U87 cells overexpressing miR-619-5p and miR-4793-3p. Transwell invasion assays were performed to investigate the effect of each miRNA and its target on GBM cell invasion. In this work, I found that two miRNAs, miR-619-5p and miR-4793-3p, are significantly downregulated in the invasive margin. Moreover, these miRNAs control the expression of genes that are involved in regulating lipid metabolism pathways. A second key finding was that the knockdown or pharmacological inhibition of the miR-619-5p target, CPT2, also significantly reduced the invasion of the cells in the invasive margin. Because CPT2 lies in the lipid metabolism pathway, my findings indicate that lipid metabolism may present a possible vulnerability of the GBM invasive cell population, and the understanding the function of miRNAs in regulating lipid homeostasis in GBM may provide novel avenues for GBM therapy.

Abstract B17: Interleukin-17 and -22 expression in non-small cell lung cancer

Abstract Background: In non-small cell lung cancer (NSCLC) the TNM staging remains standard for prognostic assessment and therapy decisions. Nevertheless, stage-specific outcomes vary significantly, indicating a need for additional prognosticators. In this retrospective study we wanted to assess the role of the mediator interleukin-22 (IL-22), as IL-22 is known to be involved in various lung diseases and to be elevated in lung cancer patients. Moreover, we evaluated interleukin-17 (IL-17), which also depicts a proinflammatory cytokine, but seems to play a dual role in antitumor immunity. We asked the question of their link to prognosis, therapy response and recurrence. Methods: Tissue microarrays (TMAs) were generated from formalin-fixed, paraffin embedded tissue of 138 curatively resected patients with stage IA-IV NSCLC. TMAs included each three cores from the tumor center (CT) and invasive margin (IM), selected from areas with the most dense lymphocytic infiltrates. IL-22 and IL-17 expression of the tissue was analyzed by immunohistochemistry via cytoplasmic staining (double-staining: IL-22, CD3 resp. IL-17, CD3). Results: IL-22 expression status in adeno and SCC does not seem to have a prognostic impact on survival, regardless of the localization. However, a high IL-22 CT/IM ratio in adenocarcinoma is clearly linked to longer overall survival; this cannot be seen in SCC. For IL-17 a distinct tendency is visible for adeno and SCC in the invasive margin as well as in the tumor center—interestingly, this is more clear for SCC. In SCC a high CT/IM ratio for IL-17 seems to have a positive prognostic impact on overall survival. Patient numbers will be enlarged to validate these findings. Conclusion: The cytokines IL-22 and IL-17 seem—depending on histology and location—to have a prognostic impact on overall survival in NSCLC. In previous experiments we showed that multispectral assessment of CD8 and PD-L1 has a clear correlation with clinical outcome. Adding cytokines to this method might open novel avenues for predicting clinical outcome and therapeutic efficacy in NSCLC. Citation Format: Julia Stump, Yue Jiang, Clara Karches, Javier Suárez Gosálvez, Jens Neumann, Sebastian Kobold, Rudolf Hatz, Hauke Winter, Amanda Tufman, Rudolf M. Huber, Simone Reu. Interleukin-17 and -22 expression in non-small cell lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B17.

Imaging Tiny Hepatic Tumor Xenografts via Endoglin-Targeted Paramagnetic/Optical Nanoprobe.

Surgery is the mainstay for treating hepatocellular carcinoma (HCC). However, it is a great challenge for surgeons to identify HCC in its early developmental stage. The diagnostic sensitivity for a tiny HCC with a diameter less than 1.0 cm is usually as low as 10-33% for computed tomography (CT) and 29-43% for magnetic resonance imaging (MRI). Although MRI is the preferred imaging modality for detecting HCC, with its unparalleled spatial resolution for soft tissue, the commercially available contrast agent, such as Gd3+-DTPA, cannot accurately define HCC because of its short circulation lifetime and lack of tumor-targeting specificity. Endoglin (CD105), a type I membrane glycoprotein, is highly expressed both in HCC cells and in the endothelial cells of neovasculature, which are abundant at the tumor periphery. In this work, a novel single-stranded DNA oligonucleotide-based aptamer was screened by systematic evolution of ligands in an exponential enrichment assay and showed a high binding affinity ( KD = 98 pmol/L) to endoglin. Conjugating the aptamers and imaging reporters on a G5 dendrimer created an HCC-targeting nanoprobe that allowed the successful visualization of orthotopic HCC xenografts with diameters as small as 1-4 mm. Significantly, the invasive tumor margin was clearly delineated, with a tumor to normal ratio of 2.7 by near-infrared (NIR) fluorescence imaging and 2.1 by T1-weighted MRI. This multimodal nanoprobe holds promise not only for noninvasively defining tiny HCC by preoperative MRI but also for guiding tumor excision via intraoperative NIR fluorescence imaging, which will probably gain benefit for the patient's therapeutic response and improve the survival rate.

Density and location of CD3+ and CD8+ tumor-infiltrating lymphocytes correlate with prognosis of oral squamous cell carcinoma.

Tumor-infiltrating lymphocytes (TILs) are regarded as adaptive immune response of the host to cancer cells and valuable prognostic factors. Here, we sought to characterize the densities and locations of CD3+ and CD8+ TILs in primary oral squamous cell carcinoma (OSCC) samples and assess their clinicopathological and prognostic significance. A total number of 169 OSCC samples from 2 independent patient cohorts (Nanjing cohort, 93 cases; Wuxi cohort, 76 cases) were retrospectively collected. The numbers of CD3+ and CD8+ TILs at tumor center (CT) and invasive margin (IM) of OSCC were identified by immunohistochemistry and calculated. The optimal cutoff values for CD3+ and CD8+ TILs to stratify patients were determined by X-tile software in Nanjing cohort and further utilized in Wuxi cohort. The associations between CD3+ /CD8+ TILs and clinicopathological parameters or patient survival were assessed. The prognostic values of CD3+ / CD8+ TILs were evaluated by Cox regression analyses. CD3+ and CD8+ TILs were identified at both CT and IM and enriched at IM. High density of CD3+ TILs at IM (CD3 IM) was significantly associated with increased overall and disease-specific survival (P<.05). High density of CD8+ TILs at CT (CD8 CT) was significantly associated with increased overall but not disease-specific survival. Moreover, CD3 IM and CD8 CT were identified as independent prognostic factors for patient survival. Our findings provide further evidence to support the prognostic values of CD3+ and CD8+ TILs for OSCC, suggesting that TIL subsets might be viable biomarkers and therapeutic targets with translational significance.

Promoter polymorphisms in TGFB1 and IL10 genes influence tumor dendritic cells infiltration, development and prognosis of colorectal cancer.

Anti-inflammatory cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-β1 have a complex role in the development of colorectal cancer (CRC). Dendritic cells (DCs) are the cellular component of the inflammatory microenvironment in the tumor and infiltration of tumors by DCs is associated with better prognosis and fewer metastases. In the present study, we explored the role of two single nucleotide polymorphisms (SNPs) in the promoter regions of TGFB1 and IL10 genes and their associations with infiltrating DCs in CRC.A case-control study was designed. Genotyping was performed via the polymerase chain reaction-restriction fragment length polymorphism method and DC infiltration was determined immunohistochemically. For the TGFΒ1 -509C/T SNP, we found that the T allele was less frequent in patients than in controls (p = 0.031) and the TT-genotype had a 2.74-fold lower risk for CRC than the CC-genotype (odds ratio = 0.365, 95% confidence interval = 0.15-0.88, p = 0.015). Additionally, the TT carriers had the shortest median survival (14.4months) (p = 0.045). The C-allele genotypes had a significantly longer survival compared to TT carriers (p = 0.018). The CC genotype was associated with a lower cellular density of CD11c in the invasive margin of the tumor (p = 0.033), whereas there was an opposite finding for CD83+ DCs (p = 0.037). Carriers of A-allele genotypes of the IL10 -1080A/G SNP had significantly lower CD83+ cells (p = 0.046) in the tumor invasive margin. The T-allele of the TGFB1 -509C/T SNP might be a protective factor for development of CRC, although, in the course of the disease, this variant allele might be associated with more unfavorable prognosis of the patients.

Combined analysis of tumor budding and tumor microenvironment in patients with stage III colorectal cancer.

652 Background: Tumor budding (TB) represents the epithelial-mesenchymal transition (EMT) and is a novel marker that predicts metastasis and poor survival in patients with colorectal cancer. Although recent preclinical studies have elucidated the interaction between the EMT process and tumor microenvironment (TME), the clinicopathological correlation between TB and TME remains unclear. Methods: Formalin-fixed paraffin-embedded blocks of specimens were obtained from patients with stage III colorectal cancer who underwent surgical resection and adjuvant chemotherapy at our institution between January 2009 and July 2012. TB, tumor stroma percentage (TSP), and inflammatory reaction (IR) graded using the Klintrup-Mäkinen method were evaluated on hematoxylin and eosin sections. The densities of CD8+ T-cells at the tumor centers and invasive margins were analyzed using immunohistochemistry and digital image analysis. Cox proportional hazards models were used to assess the effect of clinicopathological variables on relapse-free survival (RFS). Results: One hundred and ninety-five patients were included in this analysis. The median age was 62 years (range 32–84 years). The median follow-up duration of this study was 5.8 years. High TB ( &gt; 5 buds/0.785 mm2) was observed in 106 patients (54.4%) and was associated with high TSP (P &lt; 0.01), but not with IR and CD8 expression. Multivariate analysis, including clinicopathological factors such as histology, TB, TSP, and IR revealed that high TB was an independent poor prognostic factor (hazard ratio, 1.89; 95% confidence interval, 1.04–3.45; P = 0.04). Patients with high TB and low IR (21.0%) exhibited a shorter survival than others; the 5-year RFS rates were 82.7%, 81.1%, 78.4%, and 40.8% in patients with low TB and high IR, low TB and IR, high TB and high IR, and high TB and low IR, respectively. Conclusions: Our study demonstrated that high TB was an adverse prognostic factor, regardless of TME status. The combined analysis of TB plus IR could improve prognostic value in patients with stage III colorectal cancer. Patients with high TB and low IR may need novel therapeutic approaches.

Immunoclassification characterized by CD8 and PD-L1 expression is associated with the clinical outcome of gastric cancer patients.

BackgroundGastric cancer (GC) is a major cause of cancer deaths, especially in Eastern Asia. Current classification systems, including the WHO, Lauren, and TCGA, have clarified the pathological and molecular profiles of GC. However, these classifications lack an association with clinical outcome and guidance for medication selection.ObjectiveWe aimed to identify a new immunoclassification for GC to better predict patient prognosis and aid in patient selection for immunotherapy.ResultsFor all samples, 35 were EBV positive (+) and 112 were EBV negative (-). EBV infection was associated with the number of CD3+ T cells (OR = 2.91 95% CI 1.27-6.68, p = 0.012) and PD-L1 expression in TME (OR = 2.57, 95% CI 1.13–5.82, p = 0.024). EBV+ patients showed a poor overall survival (OS) compared with EBV- patients (HR = 2.37; 95% CI, 1.03–5.41; p = 0.011). Importantly, WIR patients lived significantly shorter than SIR patients with high CD8+ T cells and low PD-L1 expression (HR = 3.37; 95% CI, 1.63–6.97; p = 0.015).Materials and Methods147 formalin-fixed and paraffin-embedded (FFPE) samples of GC were obtained. Epstein-Barr virus (EBV) infection was measured. Immune markers including CD3, CD8 and PD-L1 were detected by immunohistochemistry (IHC) at tumor infiltration area (TI) and invasive margin area (IM) in tumor microenvironment (TME). PD-L1 expression was assessed by immunoreactive score (IRS) system. For immunoclassification, patients were classified into two subgroups: strong immunoreaction (SIR) and weak immunoreaction (WIR) defined by the number of CD8+ T cells and PD-L1 expression in TI.ConclusionsIn this study, we suggest a new immunoclassification for gastric cancer which is associated with patient outcome and may provide a way to guide immunotherapy in the future.

High response rate to PD-1 blockade in desmoplastic melanomas.

Desmoplastic melanoma (DM) is a rare subtype of melanoma characterized by dense fibrous stroma, resistance to chemotherapy and a lack of actionable driver mutations, but is highly associated with ultraviolet light DNA damage.1 We analysed 60 patients with advanced DM treated with programmed cell death 1 (PD-1) or PD-1 ligand (PD-L1) blocking antibody therapy. Objective tumour responses were observed in 42 of the 60 patients (70%, 95% confidence interval 57–81%), including 19 patients (32% overall) with a complete response. Whole-exome sequencing revealed a high mutational load and frequent NF-1 mutations (14 out of 17 cases). Immunohistochemistry (IHC) analysis from 19 DM and 13 non-DM revealed a higher percentage of PD-L1 positive cells in the tumour parenchyma in DM (p = 0.04), highly associated with increased CD8 density and PD-L1 expression in the tumour invasive margin. Therefore, patients with advanced DM derive significant clinical benefit from PD-1/PD-L1 immune checkpoint blockade therapy despite being a cancer defined by its dense desmoplastic fibrous stroma. The benefit is likely derived from the high mutational burden and a frequent pre-existing adaptive immune response limited by PD-L1 expression.

Abstract B069: Characterization of inflammatory cell patterns and densities using multiplex immunohistochemistry immuno-oncology assays

Abstract Definiens and Mosaic Laboratories developed an immuno-oncology (IO) panel consisting of multiplex chromogenic IHC assays and image and data analysis solutions to determine a standardized profile of the tumor microenvironment in non-small cell lung cancer (NSCLC). Expression of PD-L1 on tumor and immune cells, the density of infiltrating immune cell populations, and their spatial pattern have been shown to be predictive for response to immunotherapy. These factors are currently being evaluated to determine whether they can guide treatment across different categories of immunotherapies (e.g., checkpoint inhibition, vaccination, CAR-T cell therapy, or combinations thereof). Multiplex chromogenic IHC assays were developed for PDL1/CD68/CD3 and FoxP3/PD-1/CD8 and single-stain IHC was used to detect cells that were positive for granzyme B. Image analysis were optimized to detect biomarker positive cells (e.g., PD-L1+, CD3-, CD68-), double positive cells (e.g., PD-L1+, CD3-, CD68+), negative cells, or percent of area positive for Granzyme B. Densities for each cell population were quantified and pathologist annotations were used to distinguish between tumor core and invasive margin regions. Machine learning with random forests and decision trees was used to automatically distinguish tumor and stroma compartments to evaluate differences in densities of infiltrating immune cells. Densities of the different cell populations and their spatial relationships were analyzed to assign cases to one of the following immune status categories: acquired immune resistance, immune tolerance, adaptive checkpoint inhibitor resistance (all three are subtypes of high immune cell density), impaired infiltration, immune ignorance, intrinsic checkpoint inhibitor resistance (subtypes of low immune cell density). NSCLC tissues were analyzed and examples of biomarker profiles for each category are shown. The distribution of immune categories between squamous and non-squamous NSCLC subtypes and a comparison of immune cell densities and spatial relationships are shown. We conclude that the IO panel can be used to establish standardized immune profiles in NCCLC. In the future, it can be used to search for novel prognostic and predictive signatures and to establish the prevalence of patients with defined biomarker profiles in different subsets of patients (e.g, different stages, morphologic subtypes, or mutational status). Citation Format: Lisa M. Dauffenbach, Christopher A. Kerfoot, Gela Sia, Anthony Masci, Johannes Zimmermann, Jan Lesniak, Alexei Budco, Svenja Lippok, Katrin Schneider, René Korn, Tobias Wiestler, Dasa Medrikova, Florian Leiß. Characterization of inflammatory cell patterns and densities using multiplex immunohistochemistry immuno-oncology assays [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B069.

Second harmonic generation for collagen I characterization in rectal cancer patients with and without preoperative radiotherapy

Rectal cancer is treated with preoperative radiotherapy (RT) to downstage the tumor, reduce local recurrence, and improve patient survival. Still, the treatment outcome varies significantly and new biomarkers are desired. Collagen I (Col-I) is a potential biomarker, which can be visualized label-free by second harmonic generation (SHG). Here, we used SHG to identify Col-I changes induced by RT in surgical tissue, with the aim to evaluate the clinical significance of RT-induced Col-I changes. First, we established a procedure for quantitative evaluation of Col-I by SHG in CDX2-stained tissue sections. Next, we evaluated Col-I properties in material from 31 non-RT and 29 RT rectal cancer patients. We discovered that the Col-I intensity and anisotropy were higher in the tumor invasive margin than in the inner tumor and normal mucosa, and RT increased and decreased the intensity in inner tumor and normal mucosa, respectively. Furthermore, higher Col-I intensity in the inner tumor was related to increased distant recurrence in the non-RT group but to longer survival in the RT group. In conclusion, we present a new application of SHG for quantitative analysis of Col-I in surgical material, and the first data suggest Col-I intensity as a putative prognostic biomarker in rectal cancer.

Abstract 4567: Immunoclassification of gastric cancer in the context of clinical outcome

Abstract Background: Gastric cancer (GC) plays a leading role in all cancer deaths especially in Eastern Asia. Current classifications including WHO, Lauren, and TCGA defined molecular classification have illuminated the clinicopathological characteristics or genetic profile of GC. However, these classifications were lack of association with clinical outcome and guidance for medication selection. Objective: We aimed to identify a new immunoclassification for GC to predict patient’s prognosis and provide evidence for choosing proper medication. Methods: Formalin-fixed and paraffin-embedded (FFPE) samples of GC were obtained along with the clinical outcome of the patient. Epstein-Barr virus (EBV) infection was measured by RT-PCR. Immune markers including CD3, CD8 and PD-L1 were measured by immunohistochemistry (IHC) at tumor infiltration area (TI) and invasive margin area (IM). The expression of PD-L1 in tumor microenvironment (TME) was assessed by immune reactive score (IRS) system. For immunoclassification, patients were classified into two subgroups: strong immunoreaction (SIR) and weak immunoreaction (WIR) defined by the number of CD8+ T cells and PD-L1 expression at TI. Results: EBV infection was associated with the number of CD3+T cells and PD-L1 expression in TME. EBV+ patients also showed a poor overall survival (OS) compared with EBV- patients. Importantly, WIR patients lived significantly shorter than SIR patients. Moreover, patients who were treated by taxanes in WIR group had a shorter OS compared with those not using taxanes. Conclusion: In this study, we suggest a new immunoclassification for gastric cancer which is associated with patients’ outcome and may provide a way to guide chemotherapy. Citation Format: Weili Wang, Ping Liao, Shangchen Xie, Dongya Shen, Chengfang Zhou, Howard Mcleod, Yijing He. Immunoclassification of gastric cancer in the context of clinical outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4567. doi:10.1158/1538-7445.AM2017-4567

Programmed cell death-ligand 1 expression and immunoscore in stage II and III non-small cell lung cancer patients receiving adjuvant chemotherapy.

Programmed cell death 1 (PD-1) receptor–ligand interaction is a major pathway that is often hijacked by tumors to suppress immune control. Immunoscore (IS), a combinational index of CD3 and CD8 tumor-infiltrating lymphocyte (TIL) density in the tumor’s center and invasive margin, is a new prognostic tool suggested to be superior to conventional tumor-staging methods in various tumors. This retrospective study aimed to investigate the prevalence and prognostic roles of PD-ligand 1 (PD-L1) expression and IS in non-small cell lung cancer (NSCLC) patients receiving adjuvant chemotherapy. PD-L1 expression and TIL density were evaluated by immunohistochemical analysis in 36 patients with stage II and III NSCLC. Tumors with staining in over 1% of their cells were scored as positive for PD-L1 expression, and we determined the median number of CD3- and CD8-positive TILs as the cutoff point for TIL density. To determine IS, each patient was given a binary score (0 for low and 1 for high) for CD3 and CD8 density in both the tumor center and invasive margin region. PD-L1 expression in tumor cells was observed in 61.1% (22/36) of patients. PD-L1 expression was significantly associated with high IS, and highest IS tended to have a favorable disease-free survival.

Analysis of mutational burden and adaptive immune response in desmoplastic melanomas treated with PD-1/L1 inhibitors.

9558 Background: Desmoplastic melanoma (DM) is a rare subtype of melanoma characterized by a dense fibrous stroma, resistance to chemotherapy and no actionable driver mutation for targeted therapy. We investigated the efficacy of PD-1/L1 inhibitors and correlation with genetic landscape and tumor immune microenvironment in DM. Methods: Retrospective analysis of 1054 pts with melanoma treated with anti-PD-1/L1, resulting in 57 pts with unresectable or metastatic DM. Available baseline biopsies were analysed by digital quantitative immunohistochemistry (IHC) for CD8 and PD-L1 and by whole exome sequencing (WES), compared to available tissue from non-DM pts treated with anti-PD1/L1 at UCLA. Results: At a median follow up of 20 mo, 40 pts (70%, 95% CI 57-82) had an objective response by RECIST 1.1 criteria, including 18 (45%) CRs with no relapse observed to date. Responses were similar in DM subsets (23 pure, 29 mixed and 5 indeterminate). Kaplan-Meier estimated 1-year and 2-year overall survival were 85% (95% CI 78-98) and 74% (95% CI 64-89). WES revealed a median of 1282 (interquartile range 517-1692) non-synonymous somatic mutations per tumor in DM tumors (n = 17), significantly higher (p = 0.02) than the median of 462 (interquartile range 230-1150) in non-DM (n = 23). Mutations in NF-1 were the most common (13/17) followed by loss-of-function TP53 and ARID2, and &gt; 82% of single nucleotide mutations were UV damage signatures. IHC analysis from 19 DM and 13 non-DM revealed a strikingly higher percentage of PD-L1 positive cells in the tumor parenchyma in DM (p = 0.04), highly associated with CD8 density and PD-L1 expression in the tumor invasive margins, indicating an active adaptive immune response. No genetic mechanisms known to cause constitutive PD-L1 expression were detected in these samples. Conclusions: Patients with advanced DM derive significant clinical benefit from PD-1/L1 inhibitors, likely related to the high mutational burden and a highly active adaptive immune response as the main mechanism of immune escape prior to therapy. Our results challenge the general conception that dense fibrous stroma around the malignant cells interferes with immune cell infiltration and efficacy of immunotherapy.

Exploratory biomarker analysis in avelumab-treated patients with metastatic Merkel cell carcinoma progressed after chemotherapy.

9557 Background: Merkel cell carcinoma (MCC) is a rare but aggressive skin cancer. Tumor oncogenesis is linked to Merkel cell polyomavirus (MCPyV) integration and UV exposure. PD-L1 is often expressed in MCC tumors, suggesting that patients with MCC could benefit from anti-PD-L1 therapy. Avelumab is a fully human anti-PD-L1 IgG1 monoclonal antibody that has demonstrated clinical efficacy in patients (pts) with metastatic MCC (mMCC) in a Phase 2 trial with an objective response rate (ORR) of 31.8% in the primary analysis. Assessment of candidate predictive biomarkers may help to identify patients with a greater probability of response to avelumab and to improve understanding of MCC biology. Methods: Patients in a Phase 2 trial (NCT02155647) with mMCC and tumor progression on prior chemotherapy received avelumab at 10 mg/kg Q2W. PD-L1 expression, MCPyV status and CD8+ T-cell infiltration in pretreatment tumor samples were evaluated by immunohistochemistry (IHC). MCPyV status was also evaluated by real-time PCR. Results: Tumor PD-L1 expression was evaluable in 74 of 88 (84.1%) pts with mMCC treated with avelumab, of which 58 (65.9%) and 19 (21.6%) pts were positive at 1% and 5% cut-offs. ORR was 34.5% and 18.8% for PD-L1 positive and negative pts at 1% cutoff, and 52.6% and 23.6% for PD-L1 positive and negative pts at 5% cutoff. MCPyV status was positive in 60% (46/77) pts evaluable by IHC and 63% (45/71) pts evaluable by PCR; of 66 pts tested by both IHC and PCR, concordance was 90.9%. MCPyV+ and MCPyV– pts had similar frequencies of PD-L1+ tumors (80% and 73%) with an ORR of 26.1% and 35.5% respectively. Baseline CD8+ T-cell infiltration was assessed at tumor invasive margin and tumor center in 53 pts, ORR was 44.4% vs 19.2% and 32.1% vs 28% for pts with high or low CD8+ T-cell density at respective locations. Conclusions: In an international cohort of pts with mMCC, avelumab had clinical activity among biomarker subgroups analyzed, including PD-L1 expression, MCPyV status and density of CD8+ tumor-infiltrating T-cells. The current biomarkers were not predictive of response but further research into understanding how avelumab mediates anti-tumor activity in MCC may identify novel biomarkers. Clinical trial information: NCT02155647.

Characterization of tumor-associated T-lymphocyte subsets and immune checkpoint molecules in head and neck squamous cell carcinoma.

The composition of tumor-infiltrating lymphocytes (TIL) reflects biology and immunogenicity of cancer. Here, we characterize T-cell subsets and expression of immune checkpoint molecules in head and neck squamous cell carcinoma (HNSCC). We analyzed TIL subsets in primary tumors (n = 34), blood (peripheral blood mononuclear cells (PBMC); n = 34) and non-cancerous mucosa (n = 7) of 34 treatment-naïve HNSCC patients and PBMC of 15 healthy controls. Flow cytometry analyses revealed a highly variable T-cell infiltration mainly of an effector memory phenotype (CD45RA−/CCR7−). Naïve T cells (CD45RA+/CCR7+) were decreased in the microenvironment compared to PBMC of patients, while regulatory T cells (CD4+/CD25+/CD127low and CD4+/CD39+) were elevated. Furthermore, we performed digital image analyses of entire cross sections of HNSCC to define the ‘Immunoscore’ (CD3+ and CD8+ cell infiltration in tumor core and invasive margin) and quantified MHC class I expression on tumor cells by immunohistochemistry. Immune checkpoint molecules cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1) were increased in TILs compared to peripheral T cells in flow-cytometric analysis. Human papillomavirus (HPV) positive tumors showed higher numbers of TILs, but a similar composition of T-cell subsets and checkpoint molecule expression compared to HPV negative tumors. Taken together, the tumor microenvironment of HNSCC is characterized by a strong infiltration of regulatory T cells and high checkpoint molecule expression on T-cell subsets. In view of increasingly used immunotherapies, a detailed knowledge of TILs and checkpoint molecule expression on TILs is of high translational relevance.

Tumor-infiltrating immune cells and prognosis in gastric cancer: a systematic review and meta-analysis.

Tumor-infiltrating immune cells are a pivotal component of the tumor microenvironment (TME), but their indicative role remains poorly defined. A meta-analysis was performed to reveal the prognostic efficiency of tumor-infiltrating immune cells in gastric cancer (GC). By searching PubMed and Embase, we identified a total of 35 eligible articles that involved 4888 patients. Random or fixed effect models were employed to extract pooled hazard ratios (HRs) with 95% confidence intervals (CIs). Our results indicated that high CD3+ lymphocyte infiltration in all the locations (AG), the tumor nest (TN), and the tumor stroma (TS) predicted better overall survival (OS) (HR=0.71, 95% CI=0.57-0.90; HR=0.58, 95% CI=0.42-0.80; and HR=0.50, 95% CI=0.37-0.68, respectively). CD8+ T cell infiltration in AG and FoxP3+ regulatory T cells (Tregs) in the tumor invasive margin (TM) were also associated with improved OS (HR=0.90, 95% CI=0.83-0.97; HR=0.65, 95% CI=0.48-0.87, respectively). However, contrasting results were found in the macrophage subset, with M2 in AG (HR=1.45, 95% CI=1.13-1.86) and the TN (HR=1.67, 95% CI=1.12-2.48) associated with worse OS. In summary, the combination of the densities and locations of tumor-infiltrating immune cells can be useful for predicting survival for GC patients, but additional research is needed to reinforce the reliability of this study’s conclusions.