Supplementary Data - materials and methods

Although cancer immunotherapy approaches have focused primarily on driving T cell mediated immunity, attention has turned to other immune cell types within the tumor microenvironment (TME) to improve efficacy. Growing evidence supports a role for tumor-infiltrating B cells in complementing T cell-mediated immunity and contributing to the immunomodulation of cancer. Analyses of RNA sequencing data from The Cancer Genome Atlas (TCGA) have correlated high expression of B cell genes with improved patient survival in a range of cancer types. However, it is likely that effective anti-tumor immunity involves interactions between both B and T cells. To investigate the relative contribution of B cells to the overall TME across multiple tumor types simultaneously, we have examined a multi-tumor tissue microarray (TMA) comprising 29 different cancer types represented by approximately 12 unique donors per indication and duplicate 1mm cores per donor (1 from invasive margin [IM] and 1 from tumor center [TC]). Serial sections were stained by single-plex immunohistochemistry for CD20, CD3, CD4 and CD8, and immune cells enumerated by digital image analysis (CellProfilerTM). Analysis of overall immune infiltration regardless of core location (IM and TC combined) revealed tumor indications such as NSCLC, gastric, TNBC, cutaneous SCC and cervical to be most highly infiltrated, and GIST, GBM and prostate to exhibit the fewest tumor-infiltrating immune cells. B cell frequencies correlated with T cell densities for most tumor types with some notable exceptions including, for example, ER+ BC and gallbladder cancer where the proportion of B cells was higher relative to T cells, and TNBC where the converse pattern was evident. Spatial analyses revealed increased B cell frequencies in IM compared with TC, correlating with the distribution of T cells in the TME for the majority of tumor indications. However, a contrasting expression pattern was observed for ER+ BC, NSCLC SCC and cutaneous SCC where B cell frequencies were greater in TC. Clear evidence for presence of B cells in tertiary lymphoid structures (TLS) was observed for some tumor donor cores (e.g. gastric cancer). Taken together, we describe the relative distribution of B cells in the TME across multiple tumor types simultaneously. This approach demonstrates the benefits of utilizing an immuno-oncology focussed multi-tumor TMA to interrogate B cell biology and to dissect how heterogeneity of this cell population may contribute to anti-tumor immunity. Citation Format: Milan Bhagat, Leorenzo Memeo, Christopher Womack, Woo Hoo Kim, Marie Cumberbatch. Relative contribution of tumor infiltrating B cells to the tumor microenvironment assessed using an immuno-oncology focussed multi-tumor tissue microarray. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4646.

Supplementary Table 1. Patient characteristics

Supplemental Table 1: Design of Affymetrix and NanoString probes Supplemental Table 2: Genes included in the NanoString Codeset, including genes up- and down-regulated by MEK, Cres and KRAS signatures, plus housekeeper genes Supplemental Table 3: Correlation between Affymetrix and NanoString data for Batch Comparison or New probes Supplemental Table 4: Pearson's correlation coefficients for MEK and Cres gene probes in NSCLC FFPET Supplemental Fig 1: The MEK signature is dynamic in lung cancer cell lines as demonstrated by MEK inhibition with selumetinib and siRNA mediated KRAS knockdown Supplemental Fig 2: Pearson's correlation coefficients for the most correlated MEK and Cres probes in NSCLC FFPET Supplemental Fig 3: NanoString gene expression range of MEK and CRes genes Supplemental Fig 4: Scatterplots showing correlations between MEK, Cres and RAS signatures Supplemental Fig 5: Pearson's correlations demonstrated a high agreement of MEK signature expression between gene probe batches

2620 Background: Programmed death ligand-1 (PD-L1) contributes to immune suppression in the tumor microenvironment (TME) by interacting with programmed cell death-1 (PD-1) on infiltrating T lymphocytes leading to tumor immune escape. Application of omics technologies has shed light on the relevance of the TME for response to immunotherapies and development of novel treatment options. Here we have applied a multi-plex immunofluorescence/multi-tumor tissue microarray (TMA) approach to examine the immunobiology of different TMEs with respect to the contribution by tumor cells and macrophages to overall PD-L1 expression. Methods: A TMA comprising 11 tumor types and a total of 144 different donors, each represented by two cores [1mm; 1 from invasive margin (IM) and 1 from tumor center (TC)], was stained using Ultivue’s Immuno8 FixVUE panel (CD3, CD4, CD8, FOXP3, CD68, PD-1, PD-L1, pan-CK/SOX10). Whole slide images from two rounds of imaging (x20 magnification; four markers in each round) were aligned using Ultivue's UltiStacker software based on the nuclear counterstains from the two imaging rounds, to provide precise marker colocalization data. Cell phenotype data for each core was generated using Visiopharm software. Results: Overall PD-L1 positivity was greatest for NSCLC, SCLC, TNBC and gastric cancer ranging from approximately 600-1000 PD-L1+ cells/mm2/core, compared with CRC, breast, pancreatic, liver, and gastric esophageal junction (GOJ) cancers (approx. 50-300 PD-L1+ cells/mm2/core). Contribution by macrophages to overall PD-L1 expression (dual CD68+/PD-L1+) varied by tumor type representing 25-35% for NSCLC (SCC and ADC) and gastric cancer, whereas a converse pattern was apparent for SCLC, TNBC, breast (ER+ and Her2+) and pancreatic cancers where PD-L1+ macrophages accounted for a large proportion (approx. 60-85%) of overall tumor PD-L1 expression. Interestingly, as a proportion of total macrophage infiltration, approximately 65% of CD68+ macrophages were PD-L1+ for SCLC and TNBC, compared with less than 10% for pancreatic and liver cancer. Spatial analysis revealed PD-L1+ macrophage infiltration to be generally higher for IM versus TC, and the distribution between tumor (CK+) and stroma (CK-) remained similar despite exclusion of CD3+/CD8+ cytotoxic T cells from CK+ tumor regions for pancreatic and liver cancer. Conclusions: Taken together, these data illustrate the benefits of combining multiplexed immunofluorescence staining, with digital analysis of cell phenotypes within well characterized tumor samples to better understand the relative immunobiology of different TMEs. Here we demonstrate that the relative contribution of macrophages to the overall PD-L1 microenvironment varies between tumor types, which may help guide options for successful immunotherapy strategies.

Abstract The tumor microenvironment (TME) represents a complex interaction of immune and host cells and can be described broadly as ‘inflamed', ‘immune excluded' and ‘desert'; categories that may serve as predictive parameters for response to different immunotherapies. Multiplexed fluorescence and associated digital imaging platforms are important tools for defining the spatial components of the TME and can accelerate development of novel immunotherapy strategies. To investigate the TME across multiple tumor types simultaneously we have utilized a multi-tumor tissue microarray (TMA) covering 11 different tumor types comprising a total of 144 cases, each in duplicate with cores (1mm) taken from invasive margin (IM) and tumor center (TC). The TMA was stained using the UltiMapper I/O Immuno8 panel, which includes markers for CD3, CD4, CD8, FOXP3, CD68, PD-1, PD-L1, and a pan-CK/SOX10 cocktail as a tumor indicator. Stained TMAs were scanned on a fluorescence whole slide scanner (20X magnification) and marker images aligned using the UltiStacker software using the nuclear counterstain images as references from two rounds of imaging, to provide accurate marker colocalization data. Digital image analysis was performed using Visiopharm software to generate cell phenotype data, including localization to tumor and stroma, for each of the 288 cores. Comparative analysis of all tumor types regardless of core location (IM and TC combined) revealed gastric cancer, CRC, TNBC and NSCLC to be most highly infiltrated with CD3+ T cells, and ER+ and Her2+ breast cancers least infiltrated. The contribution of cytotoxic T cells (CD3+ CD8+) to the TME was greater for gastric cancer (approx. 50%) compared with NSCLC (approx. 20%) and the proportion of cytotoxic T cells activated/exhausted as defined by expression of PD-1 ranged from approx. 25% (eg. TNBC, CRC) to 1% (eg. ER+ BC). Spatial localization analyses of cytotoxic T cells revealed pancreatic and hepatic cancers as the most ‘excluded' as evidenced by low CD3+/CD8+ T cell numbers in tumor (CK+) compared with stromal regions (CK-). Interestingly, cytotoxic T cells with an exhausted phenotype (CD3+ CD8+ PD-1+) were preferentially infiltrated within CK+ tumor areas compared with stroma (CK-) for inflamed tumors such as gastric, TNBC and CRC. These data highlight the benefits of combining a fast, multiplexed staining protocol, with a detailed digital analysis of immune phenotypes spatially within well characterized tumor samples to better understand the TME (eg. inflamed, excluded) and identify tumor types more likely to benefit from different immunotherapeutic strategies. Citation Format: Marie Cumberbatch, Douglas Wood, Christopher Womack, Milan Bhagat, Mael Manesse, Alison Bigley, Lorcan Sherry. Comparative multiplex digital phenotyping of the tumor microenvironment across multiple tumor types using a well characterized multi-tumor tissue microarray [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2778.

Abstract Macrophages contribute to cancer-associated inflammation, and those with M2-like phenotype have been reported to be involved in tumor progression, immunosuppression, metastasis and angiogenesis. CD163 is considered a marker of M2-like macrophages/monocytes and high expression has been associated with poor prognosis in various tumor indications. To investigate the relative contribution of macrophage/monocyte cell populations to the tumor immune microenvironment across multiple tumor types concurrently, we have utilised a multi-tumor tissue microarray (TMA) comprising 30 different tumor indications, with approximately 12 cases per tumor type, each case represented by duplicate 1mm cores (1 from IM and 1 from TC). Serial sections of TMA slides (n=5) were stained by immunohistochemistry for CD68 and CD163, and cells/mm2/core delivered by digital image analysis of scanned images (CellProfilerTM). Comparative analysis of all tumor types regardless of core location (IM and TC combined) revealed lung, renal and cervical cancers to be the most highly infiltrated with CD68+ macrophages, while the greatest CD163 staining was observed for sarcoma and glioblastoma (GBM). Interestingly, a direct comparison of multiple tumor types simultaneously as performed here has revealed strongly opposing immune microenvironments in different tumor indications with respect to the balance of expression of CD68 and CD163. Higher frequencies of CD68+ macrophages relative to low CD163 expression was observed for prostate, endometrial, renal, cervical, thyroid, pancreatic, hepatic, small bowel and urothelial cancers. Conversely, a strong M2-like milieu exhibiting CD163+ cell frequencies exceeding CD68+ macrophages numbers was apparent for sarcoma, GBM, mesothelioma, bladder, cutaneous squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cell carcinoma, ER+ and Her2+ breast cancers. The remaining tumors, including cholangiocarcinoma, triple negative breast, lung, ovarian, gallbladder, colorectal, gastric, and esophageal cancers, all exhibited equivalent levels of infiltrating CD68+ and CD163+ cells. Analyses of IM versus TC revealed further complexities of the tumor immune microenvironment. For some disease settings, divergent CD163:CD68 ratios were observed for IM versus TC, whereas for other indications the ratio remained unchanged. In conclusion, concurrent analysis of multiple tumor indications for CD68 and CD163 has revealed immunosuppressive M2-like macrophage/monocyte tumor immune microenvironments that vary markedly between tumor types and within some tumors. These data may help to identify those patients that may benefit from therapies that target this immune pathway. Citation Format: Marie Cumberbatch, Woo Ho Kim, Lorenzo Memeo, Lorenzo Colarossi, Christopher Womack, Milan Bhagat. Opposing CD68/CD163 tumour immune microenvironments revealed using a large multi-tumor tissue microarray (TMA) comprising cores from invasive margin (IM) and tumor center (TC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3878.

Conclusions In this study, we highlighted the benefits of using a combination of well-characterized TMAs, a fast, optimized 8-plex mIHC protocol, and a detailed analysis pipeline to characterize the immune-response in a broad range of cancer types and samples, leading to a better understanding of the TME as well as a streamlined workflow for further translational studies. Results Immune cell counts and phenotypes were identified using automated analysis for cores within the tumor and within the tumor margin using a panel characterizing a range of immune cell populations, and compared across each tissue type. Deep phenotyping was performed for each core to identify unique profiles for each tissue type, with a workflow optimized for high-throughput analysis of rich-content TMAs. Methods Each slide comprised 144 cores (1 mm) and included duplicate cores for each case (1 from invasive margin; 1 from tumor center) from 11 different tumor types including breast cancer (ER+, Her2+, TNBC), NSCLC (squamous, adenocarcinoma), SCLC, CRC, pancreatic, gastric, hepatic and esophageal cancers. TMA sections were stained using the UltiMapper I/O Immuno8 panel, which includes markers for CD3, CD4, CD8, FOXP3, CD68, PD-1, PD-L1, and a pan-CK/SOX10 cocktail as a tumor indicator. The stained TMAs were scanned at 20X magnification on a fluorescence whole slide scanner. To provide accurate marker colocalization data, marker images were aligned using the UltiStacker software, using the nuclear counterstain images as references from multiple rounds of imaging. Image analysis was performed using Visiopharm software and generated total and negative cell phenotype counts, cell density in tumor and stroma, as well as spatial interactions maps in each of the 288 cores in the TMA set. Background Multiplex immunohistochemistry (mIHC) and associated data analysis methods are rapidly becoming invaluable tools to improve our understanding of the complex tumor micro-environment (TME) and accelerate the discovery of novel immunotherapy targets. These techniques can enable the accurate phenotyping of the immune response and checkpoint expression in the spatial context of the tumor. The goal of this study was to identify the populations of immune cells (T-cytotoxic, T-helper, T-reg, and macrophages), their functional status, as well as their interactions with the tumor, in a range of samples and indications using a carefully designed multi-tumor Tissue Micro-Array (TMA) set of 2 slides from TriStar.

49 Background: Archival specimens collected months or years prior to starting immunotherapy are often used to identify patients for second line immune checkpoint inhibitor (ICI) treatment. PD-L1 expression and the immune microenvironment in these patients may have altered over time following multiple lines of failed standard of care (SOC) treatments. Methods: Formalin fixed paraffin embedded (FFPE) tumor samples, taken during resection performed as first line surgical treatment from a cohort of NSCLC patients (n = 18), were evaluated by Nanostring using the IO360 gene expression panel, and by immunohistochemistry (IHC) for CD3, CD8, PD-L1, CD68 and CD163. The resultant immune profiles were correlated with the clinical follow-up data for radiotherapy, SOC chemotherapy, and second line immunotherapy with the aim of understanding whether immune signatures predictive of response to ICI therapy may be identified in such samples. Results: Of the 18 cases, clinical follow-up data indicated objective response to ICI therapy for 4 patients, with the mean time from initial diagnosis to ICI treatment being 2.8 years (range: 0.4 to 8.5 years). Although pathologist PD-L1 IHC scores were not predictive of response, IHC image analysis data revealed significant increases in CD3 (2.3-fold) and CD8 (2.7-fold) T cell numbers in the responder population. In addition, although CD68+ macrophage frequencies did not differ significantly between responder and non-responder populations, reduced M2-like CD163+ macrophage/monocyte numbers were evident for responders. While the Tumor Inflammation Signature was not predictive of response, several gene expression signatures were significantly associated with response including increased abundance of CD8 T cells, cytotoxic cells, cytotoxicity, MHC class II antigen presentation and Melanoma-Associated Antigens (MAGE). Conclusions: Despite these patients having received various lines of radiotherapy and SOC chemotherapy prior to receiving immunotherapy, immune profiles associated with response to second line immunotherapy were detected in surgical first line resection samples.

Abstract Surgical first line resection samples are frequently accessed to select patients for immune checkpoint inhibitor (ICI) therapy, often based on expression of PD-L1 measured by immunohistochemistry (IHC). However, while PD-L1 expression may enrich for response to ICIs, other immune parameters in the tumor microenvironment will likely contribute to outcome. To assist in the identification of biomarker(s) that might predict response to ICIs, we have analyzed a cohort of pre-treatment NSCLC cases for which second line immunotherapy clinical follow-up data are available. Formalin fixed paraffin embedded (FFPE) tumor samples were analysed (i) by single-plex IHC for CD3, CD8, CD68, CD163 and PD-L1, plus digital image analysis (CellProfilerTM), and (ii) profiled for multidimensional biomarkers using the targeted RNA sequencing and machine-learning platform ImmunoPrism® from Cofactor Genomics. Clinical follow-up data indicated objective response to ICI therapy for 4/18 patients, with average time from initial diagnosis to ICI treatment of 33.5 ± 29.6 months (mean ± SD). While CD68+ macrophage frequencies evaluated by IHC did not differ significantly between responder and non-responder populations, significant increases in T cell numbers (CD3: 2.3-fold; CD8: 2.7-fold; both p<0.05) were observed for the responder population. CD8 T cells were orthogonally measured using the ImmunoPrism assay, and the same significant differences for CD8 were observed. Although a trend towards decreased M2-like CD163+ macrophage/monocyte numbers was apparent by IHC for responders, this was not supported by the Cofactor analysis. When sections from the same FFPE block are analyzed across multiple commercially available platforms, there is high confidence for those signals which show concordance between platforms, such as the increase in CD8 T cell abundance observed for responders in this study. Moving beyond single-analyte biomarkers, a multidimensional biomarker combining immune escape genes and RNA-based immune cell measurements was generated using the ImmunoPrism platform with standard parameters. The resulting biomarker had the following performance characteristics: predictive accuracy, 89%; positive predictive value (PPV), 100%; negative predictive value (NPV), 88%; sensitivity, 50%; and specificity, 100%. A receiver-operating characteristic (ROC) curve was also generated for this putative biomarker, with an area under the curve (AUC) of 0.87. The promising results from this exploratory sample set warrant further investigation in a larger cohort. These data also demonstrate that clinical archives with well-curated demographic and outcome data, such as the FFPE samples analyzed here, provide excellent cohorts for biomarker screening and discovery studies. The application of new multianalyte approaches enables additional signals from the tumor microenvironment to be captured and included for predicting patient response. Citation Format: Milan Bhagat, Woo Ho Kim, Lorenzo Memeo, Lorenzo Colarossi, Natalie LaFranzo, Steve Daniel, Christopher Womack, Marie Cumberbatch. Immune biomarkers in the tumor microenvironment associated with response in pre-treatment non-small cell lung cancer (NSCLC) samples with second line immunotherapy follow-up data [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3194.

Abstract Purpose: To develop a clinically viable gene expression assay to measure RAS/RAF/MEK/ERK (RAS–ERK) pathway output suitable for hypothesis testing in non–small cell lung cancer (NSCLC) clinical studies. Experimental Design: A published MEK functional activation signature (MEK signature) that measures RAS–ERK functional output was optimized for NSCLC in silico. NanoString assays were developed for the NSCLC optimized MEK signature and the 147-gene RAS signature. First, platform transfer from Affymetrix to NanoString, and signature modulation following treatment with KRAS siRNA and MEK inhibitor, were investigated in cell lines. Second, the association of the signatures with KRAS mutation status, dynamic range, technical reproducibility, and spatial and temporal variation was investigated in NSCLC formalin-fixed paraffin-embedded tissue (FFPET) samples. Results: We observed a strong cross-platform correlation and modulation of signatures in vitro. Technical and biological replicates showed consistent signature scores that were robust to variation in input total RNA; conservation of scores between primary and metastatic tumor was statistically significant. There were statistically significant associations between high MEK (P = 0.028) and RAS (P = 0.003) signature scores and KRAS mutation in 50 NSCLC samples. The signatures identify overlapping but distinct candidate patient populations from each other and from KRAS mutation testing. Conclusions: We developed a technically and biologically robust NanoString gene expression assay of MEK pathway output, compatible with the quantities of FFPET routinely available. The gene signatures identified a different patient population for MEK inhibitor treatment compared with KRAS mutation testing. The predictive power of the MEK signature should be studied further in clinical trials. Clin Cancer Res; 23(6); 1471–80. ©2016 AACR. See related commentary by Xue and Lito, p. 1365

AbstractPurpose: AZD2014 is a novel, oral, m-TORC 1/2 inhibitor that has shown in vitro and in vivo efficacy across a range of preclinical human cancer models.Experimental Design: A rolling six-dose escalation was performed to define an MTD (part A), and at MTD a further cohort of patients was treated to further characterize toxicities and perform pre- and posttreatment biopsies (part B). AZD2014 was administered orally twice a day continuously. Flow cytometry, ELISA, and immunohistochemistry were used to quantify pharmacodynamic biomarkers. Pharmacokinetic analysis was carried out by mass spectrometry.Results: A total of 56 patients were treated across a dose range of 25 to 100 mg. The MTD was 50 mg twice daily. The dose-limiting toxicities were fatigue and mucositis. At the MTD, the most common adverse events (AE) were fatigue (78%), nausea (51%), and mucositis (49%), but these were equal to or greater than grade 3 in only 5% of patients. Drug levels achieved at the MTD (AUCss 6686 ng·h/mL, Cmax ss 1,664 ng/mL) were consistent with activity in preclinical models. A reduction in p-S6 levels and Ki67 staining was observed in 8 of 8 and 5 of 9 evaluable paired biopsy samples. Partial responses were seen in a patient with pancreatic cancer and a patient with breast cancer, who were found to have a PDGFR and ERBB2 mutation, respectively.Conclusions: The recommended phase II dose for further evaluation of AZD2014 is 50 mg twice daily, and at this dose it has been possible to demonstrate pharmacologically relevant plasma concentrations, target inhibition in tumor, and clinical responses. Clin Cancer Res; 21(15); 3412–9. ©2015 AACR.

VEGF (vascular endothelial growth factor) signaling inhibitors are widely used in different cancer types; however, patient selection remains a challenge. Analyses of samples from a phase III clinical trial in metastatic colorectal cancer testing chemotherapy versus chemotherapy with the small molecule VEGF receptors inhibitor cediranib identified circulating leptin levels, BMI, and a tumor metabolic and angiogenic gene expression signature associated with improved clinical outcome in patients treated with cediranib. Patients with a glycolytic and hypoxic/angiogenic profile were associated with increased benefit from cediranib, whereas patients with a high lipogenic, oxidative phosphorylation and serine biosynthesis signature did not gain benefit. These findings translated to pre-clinical tumor xenograft models where the same metabolic gene expression profiles were associated with in vivo sensitivity to cediranib as monotherapy. These findings suggest a link between patient physiology, tumor biology, and response to antiangiogenics, which may guide patient selection for VEGF therapy in the future.

Following the success of imiquimod, agents targeting TLR7 are being progressed as potential immunotherapeutics. Stimulation of TLR7 initiates a plasmacytoid dendritic cell driven immune response which drives efficacy. In addition to a role in regulating immune cell responses, TLR7 has also been reported to play a role in regulating tumor cell function (1,2). To investigate the expression of TLR7, by both tumor cells and by tumor infiltrating immune cells, we have used immunohistochemistry (IHC). Initially, a comprehensive validation of commercially available anti-TLR7 antibodies was conducted revealing one antibody (Epitomics, 3923-1) with specificity for TLR7 in western blots of cell lysates, and by immunocytochemistry of formalin fixed paraffin embedded (FFPE) cell pellets, of HEK293 cells stably transfected with TLR7, mock-transfected or non-transfected. 3923-1 was validated further for IHC across FFPE sections of human spleen, lymph node and tonsil demonstrating expected tissue and cellular localization. A comparison of staining for TLR7 in whole sections of FFPE tumors revealed non-specific tumor staining using antibodies that failed the validation process compared with the staining pattern observed for 3923-1. Subsequently, five tissue microarrays (TMAs) comprising 18 different human tumor types (6-25 patients/tumor type, triplicate cores) and 14 normal tissues (5 donors/tissue type, duplicate cores) were stained by IHC for TLR7 expression using 3923-1. Pathologist scores for tumor cells revealed 5/18 tumor types negative for TLR7 (ovarian, glioma, thyroid, liver, renal) and 9/18 tumor types with a proportion of patients (4%-36%) exhibiting weak (1+) staining (breast, lung, colorectal, pancreatic, gastric, head & neck, melanoma, esophageal, endometrial). Furthermore, 11-17% of sarcoma, prostate and bladder tumors displayed moderate (2+) staining for TLR7, whereas corresponding normal tissue epithelium was largely negative for TLR7. Importantly, an increased density of immune infiltrates was observed in tumor tissues compared with normal tissues, and a greater proportion of the immune infiltrate in tumors was TLR7 positive. The conclusion drawn is that TLR7 may be less frequently expressed by tumor cells than reported previously and that tumors exhibit a marked TLR7 positive immune cell infiltrate. These data identify tumour types which might benefit from TLR7 therapy and may guide patient selection. 1. Grimm M et al. Eur J Cancer (2010):2849-57. 2. Cherfils-Vicini J et al. J Clin Invest (2010):1285-97. Citation Format: Nicola Haughton, Foster Emily, Christopher Womack, Simon Barry, Setsuko Yamamoto, Masashi Murata, Marie Cumberbatch. Toll-like receptor (TLR) 7 expression in the human tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3670. doi:10.1158/1538-7445.AM2014-3670

Receptor tyrosine kinase pathways are potential therapeutic targets in gastric adenocarcinoma patients. We evaluated HER2 and cMet protein expression, and FGFR2 gene amplification to assess their prognostic significance, and downstream mediators pS6 and pERK for their potential utility as pharmacodynamic biomarkers in patients with gastric adenocarcinoma. Tissue microarrays were constructed from resection samples of 184 patients who underwent surgery for gastric/gastro-oesophageal junction adenocarcinoma. Tissue cores were obtained from the tumour body (TB), luminal surface (LS) and invasive edge (IE), and immunohistochemical and fluorescence in situ hybridisation (FGFR2) analysis was performed. FGFR2 amplification was identified in 2 % of cases and associated with worse survival (P = 0.005). HER2 overexpression was observed in 10 % of cases and associated with increased survival (P = 0.041). cMet overexpression was observed in 4 % of cases and associated with worse survival (P < 0.001). On multivariate analysis, only cMet retained significance (P = 0.006). pS6 and pERK expression were observed in 73 % and 30 % of tumours, respectively, with no association with survival. HER2 (P = 0.004) and pERK (P = 0.001) expression differed between tumour regions with HER2 expression increased in the LS compared with the TB and IE. These findings confirm subpopulations in gastric adenocarcinoma with poor outcome that may benefit from specific therapeutic strategies. However, we found heterogeneous HER2, pS6 and pERK overexpression, which presents challenges for their use as predictive biomarkers in gastric biopsies. The potential downstream pharmacodynamic markers pS6 and pERK were expressed across tumour regions, providing evidence that resections and biopsies would yield comparative results in clinical trials.

Advances in the understanding of molecular pathology and thereby the mechanisms that could be amenable to therapeutic manipulation are the reason that pharmaceutical research and development is focused increasingly on measurement of molecular biomarkers in human biological samples. Obtaining direct or indirect access to sufficient samples that are fit for research purposes can be a major challenge. A biobanking infrastructure has a significant role in the acquisition, storage and usage of human biological samples and here we review some key requirements for establishing a biobank. These include ensuring; that appropriate governance mechanisms are in place, that samples available are appropriate and fit for the intended research purposes that the infrastructure is sustainable in the future and that use of the biobank assets meets the strategic aims of the host organisation. Finally we present a case study - the STRATUM project which has recently completed and through a collaborative approach involving six industry and public partners drawing on a network of experts, examined biobank policies, public attitudes to biobanking, donor consent, sample and data standards, technical requirements for a register and biobanking financial models, albeit from a UK perspective. (C) 2014 Elsevier Inc. All rights reserved.

Immunohistochemistry ( IHC ) is a core platform for the analysis of tissue samples, and there is an increasing demand for reliable and quantitative IHC ‐based tissue biomarkers in oncology clinical research and development (R&D) environments. Biomarker assay and drug development proceed in parallel. Furthermore, biomarker assay requirements change with each phase of drug development. We have therefore developed a matrix tool to enable researchers to evaluate whether a particular IHC biomarker assay is fit for purpose. Experience gained from the development of 130 IHC biomarkers, supporting a large number of oncology drug projects, was used to formulate a practical approach to IHC assay development. The resultant matrix grid and accompanying work flow incorporates 16 core decision points that link antibody and assay specificity and sensitivity, and assay performance in preclinical and clinical samples, with stages of drug development. The matrix provides a means to ensure that relevant information on an IHC assay in development is recorded and communicated consistently and that minimum assay validation requirements are met. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

We completed a phase I clinical trial to test the safety and toxicity of combined treatment with cixutumumab (anti-IGF-1R antibody) and selumetinib (MEK 1/2 inhibitor). Patients with advanced solid tumours, refractory to standard therapy received selumetinib hydrogen sulphate capsules orally twice daily, and cixutumumab intravenously on days 1 and 15 of each 28-day cycle. The study used a 3+3 design, with a dose-finding cohort followed by an expansion cohort at the maximally tolerated dose that included pharmacokinetic and pharmacodynamic correlative studies. Thirty patients were enrolled, with 16 in the dose-finding cohort and 14 in the expansion cohort. Grade 3 or greater toxicities included nausea and vomiting, anaemia, CVA, hypertension, hyperglycaemia, and ophthalmic symptoms. The maximally tolerated combination dose was 50 mg twice daily of selumetinib and 12 mg kg−1 every 2 weeks of cixutumumab. Two patients achieved a partial response (one unconfirmed), including a patient with BRAF wild-type thyroid carcinoma, and a patient with squamous cell carcinoma of the tongue, and six patients achieved time to progression of >6 months, including patients with thyroid carcinoma, colorectal carcinoma, and basal cell carcinoma. Comparison of pre- and on-treatment biopsies showed significant suppression of pERK and pS6 activity with treatment. Our study of anti-IGF-1R antibody cixutumumab and MEK 1/2 inhibitor selumetinib showed that the combination is safe and well-tolerated at these doses, with preliminary evidence of clinical benefit and pharmacodynamic evidence of target inhibition.