John Alvarez

Validation of biomarkers to predict response to immunotherapy in cancer: Volume II — clinical validation and regulatory considerations

There is growing recognition that immunotherapy is likely to significantly improve health outcomes for cancer patients in the coming years. Currently, while a subset of patients experience substantial clinical benefit in response to different immunotherapeutic approaches, the majority of patients do not but are still exposed to the significant drug toxicities. Therefore, a growing need for the development and clinical use of predictive biomarkers exists in the field of cancer immunotherapy. Predictive cancer biomarkers can be used to identify the patients who are or who are not likely to derive benefit from specific therapeutic approaches. In order to be applicable in a clinical setting, predictive biomarkers must be carefully shepherded through a step-wise, highly regulated developmental process. Volume I of this two-volume document focused on the pre-analytical and analytical phases of the biomarker development process, by providing background, examples and “good practice” recommendations. In the current Volume II, the focus is on the clinical validation, validation of clinical utility and regulatory considerations for biomarker development. Together, this two volume series is meant to provide guidance on the entire biomarker development process, with a particular focus on the unique aspects of developing immune-based biomarkers. Specifically, knowledge about the challenges to clinical validation of predictive biomarkers, which has been gained from numerous successes and failures in other contexts, will be reviewed together with statistical methodological issues related to bias and overfitting. The different trial designs used for the clinical validation of biomarkers will also be discussed, as the selection of clinical metrics and endpoints becomes critical to establish the clinical utility of the biomarker during the clinical validation phase of the biomarker development. Finally, the regulatory aspects of submission of biomarker assays to the U.S. Food and Drug Administration as well as regulatory considerations in the European Union will be covered.

Interleukin-6 is a potential therapeutic target in interleukin-6 dependent, estrogen receptor-α-positive breast cancer.

Introduction Interleukin-6 (IL-6) is an important growth factor for estrogen receptor-α (ERα)-positive breast cancer, and elevated serum IL-6 is associated with poor prognosis. Methods The role of the phosphorylated signal transducer and activator of transcription 3 pathway was investigated in ERα-positive breast cancer. A panel of cell lines was treated with exogenous IL-6. An IL-6 specific gene signature was generated by profiling ten ERα-positive breast cancer cell lines alone or following treatment with 10 ng/mL recombinant IL-6 or human marrow stromal cell-conditioned media, with or without siltuximab (a neutralizing anti-IL-6 antibody) and grown in three-dimensional tumor microenvironment-aligned cultures for 4 days, 5 days, or 6 days. The established IL-6 signature was validated against 36 human ERα-positive breast tumor samples with matched serum. A comparative MCF-7 xenograft murine model was utilized to determine the role of IL-6 in estrogen-supplemented ERα-positive breast cancer to assess the efficacy of anti-IL-6 therapy in vivo. Results In eight of nine ERα-positive breast cancer cell lines, recombinant IL-6 increased phosphorylation of tyrosine 705 of STAT3. Differential gene expression analysis identified 17 genes that could be used to determine IL-6 pathway activation by combining their expression intensity into a pathway activation score. The gene signature included a variety of genes involved in immune cell function and migration, cell growth and apoptosis, and the tumor microenvironment. Validation of the IL-6 gene signature in 36 matched human serum and ERα-positive breast tumor samples showed that patients with a high IL-6 pathway activation score were also enriched for elevated serum IL-6 (≥10 pg/mL). When human IL-6 was provided in vivo, MCF-7 cells engrafted without the need for estrogen supplementation, and addition of estrogen to IL-6 did not further enhance engraftment. Subsequently, we prophylactically treated mice at MCF-7 engraftment with siltuximab, fulvestrant, or combination therapy. Siltuximab alone was able to blunt MCF-7 engraftment. Similarly, siltuximab alone induced regressions in 90% (9/10) of tumors, which were established in the presence which were established in the presence of hMSC expressing human IL-6 and estrogen. Conclusion Given the established role for IL-6 in ERα-positive breast cancer, these data demonstrate the potential for anti-IL-6 therapeutics in breast cancer.

Oncogenic Characterization and Pharmacologic Sensitivity of Activating Fibroblast Growth Factor Receptor (FGFR) Genetic Alterations to the Selective FGFR Inhibitor Erdafitinib

Fibroblast growth factor receptor (FGFR) genetic alterations are frequently observed in cancer, suggesting that FGFR inhibition may be a promising therapy in patients harboring these lesions. Identification of predictive and pharmacodynamic biomarkers to select and monitor patients most likely to respond to FGFR inhibition will be the key to clinical development of this class of agents. Sensitivity to FGFR inhibition and correlation with FGFR pathway activation status were determined in molecularly annotated panels of cancer cell lines and xenograft models. Pathway inhibition in response to FGFR inhibitor treatment was assessed in cell lines (both in vitro and in vivo) and in samples from patients treated with the FGFR inhibitor JNJ-42756493 (erdafitinib). Frequency of FGFR aberrations was assessed in a panel of NSCLC, breast, prostate, ovarian, colorectal, and melanoma human tumor tissue samples. FGFR translocations and gene amplifications present in clinical specimens were shown to display potent transforming activity associated with constitutive pathway activation. Tumor cells expressing these FGFR activating mutants displayed sensitivity to the selective FGFR inhibitor erdafitinib and resulted in suppression of FGFR phosphorylation and downstream signal transduction. Clinically, patients receiving erdafitinib showed decreased Erk phosphorylation in tumor biopsies and elevation of serum phosphate. In a phase I study, a heavily pretreated bladder cancer patient with an FGFR3–TACC3 translocation experienced a partial response when treated with erdafitinib. This preclinical study confirmed pharmacodynamics and identified new predictive biomarkers to FGFR inhibition with erdafitinib and supports further clinical evaluation of this compound in patients with FGFR genetic alterations. Mol Cancer Ther; 16(8); 1717–26. ©2017 AACR.

Abstract 3530: The role of IL-6 in ERα+ breast cancer and potential use for Siltuximab, an anti-IL-6 antibody, in ERα+ breast cancer treatment.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Interleukin-6 (IL-6) is an important growth factor for estrogen receptor-alpha (ERα) positive breast cancer. High IL-6 serum levels are associated with poor prognosis in ERα+ breast cancer patients, and IL-6 related polymorphisms that lead to elevated IL-6 expression are associated with decreased overall survival (PMID 17261184, 12771987). Preclinical data suggest that in contrast to ERα negative breast cancer cell lines, ERα positive cell lines rarely produce autocrine IL-6, and are therefore dependent on paracrine IL6 produced by the microenvironment. Furthermore, when ERα positive cell lines are exposed to paracrine IL-6, phosphorylation of STAT3-Y705, increased growth rates and more aggressive tumor phenotypes are observed (PMID 17586727, 18974155, 19581928). We utilized 3D co-cultures and heterotypic xenograft models to investigate the ability of siltuximab, an anti-IL-6 antibody in clinical development, to attenuate paracrine IL-6 effects across a panel of 8 ERα positive breast cancer cell lines and heterotypic xenograft models. From a panel of 8 ERα postive breast cancer lines, 75% responded to recombinant human IL-6 (hIL-6) protein by phosphorylation of STAT3 (Ty705), but not AKT, MEK1/2, or ERK1/2. Siltuximab treatment blunted pSTAT3 induction in all IL-6 responsive ERα positive breast cancer lines but failed to reduce pSTAT3 phosphorylation in three ERα negative cell lines that produced autocrine IL-6. The 3D tumor growth assay demonstrated accelerated growth rates for ERα positive breast cancer lines in the presence of IL-6 or human mesenchymal stem cells (hMSC), which returned to baseline with siltuximab treatment. In addition, when hIL-6 was supplemented in vivo, the ERα positive tumor cell line, MCF-7 engrafted without the need for estrogen supplementation and tumor growthblunted with siltuximab treatment. When tumors were allowed to establish before treatment, siltuximab was able to induce tumor regressions in all treated animals (10/10). Tumor regression was associated with decreased mitotic counts in tumors. In addition to these experiments, we investigated STAT3 activation in a panel of primary patient-derived ERα positive breast cancer samples. 43% of samples demonstrated activated STAT3 as determined by Y705 phosphorylation. These same ERα positive breast cancer patients displayed an activated IL-6 network as assessed by an a priori 32-gene IL-6 network gene expression profile signature and was also associated with increased IL-6 serum levels. This activated IL-6 gene signature was also associated with ERα positive tumors and increased metastasis as assessed by positive node status in four independent breast cancer data sets. Taken together, these data suggest a key role for IL-6 in estrogen independent ERα positive breast cancer progression. Citation Format: Amy Axel, Tineke Casneuf, Peter King, JD Alvarez, Brett Hall, Kate Sasser. The role of IL-6 in ERα+ breast cancer and potential use for Siltuximab, an anti-IL-6 antibody, in ERα+ breast cancer treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3530. doi:10.1158/1538-7445.AM2013-3530

Comparison of Immunohistochemistry Assay Results with Gene Expression Profiling Methods for Diffuse Large B-Cell Lymphoma Subtype Identification in Matched Patient Samples

Background: The cell of origin (COO) in diffuse large B-cell lymphoma (DLBCL) has prognostic importance. While the COO was originally classified into germinal center B-cell (GCB) or activated B-cell (ABC) subtypes by microarray analysis, routine use was not practical. Immunohistochemistry (IHC)–based methods are widely used with varying results due to lack of standardization. Several classification methods have been developed recently; understanding the concordance between these and existing methods is essential to their practical application. Therefore, we evaluated concordance between 3 commercial assays: a standardized Hans-based IHC method and 2 gene expression profiling (GEP) methods and compared these to the accepted microarray classification.Methods: 137 DLBCL-confirmed tumor samples were evaluated using a standardized Hans-based IHC method for GCB or non-GCB subtype, by a published microarray-based assay, a digital gene expression-based Lymphoma Subtyping Test (LST) and a next-generation sequencing-based assay (EdgeSeq COO). Subtype calls from the 3 GEP methods were harmonized to “GCB” or “non-GCB” and assessed for concordance.Results: Concordance between the Hans-based IHC assay and the microarray-based assay, LST assay and EdgeSeq assay was 79.6% (N=137), 80.0% (N=125) and 78.2% (N=64), respectively; the positive percent agreement (PPA) in non-GCB was 88.9%, 87.0% and 78.1%, respectively. Concordance for the Hans-based IHC assay versus GEP methods was especially high for direct GCB calls (91.0%, 88.3% and 78.1% for microarray, LST and EdgeSeq COO methods, respectively). The newly developed GEP assays performed well against the microarray GEP method, against which they were calibrated (concordance 93.7% and 87.5% and PPA 94.3% and 92.9%, respectively, for LST and EdgeSeq COO).Conclusion: These results demonstrated good consistency between various platforms for stratification of DLBCL into COO subtype classifications. Application of a standardized Hans-based IHC assay offers a robust, rapid and easily accessible platform to classify DLBCL into prognostically important subtypes.

Integrative analysis reveals CD38 as a therapeutic target for plasma cell-rich pre-disease and established rheumatoid arthritis and systemic lupus erythematosus

BackgroundPlasmablasts and plasma cells play a key role in many autoimmune diseases, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). This study was undertaken to evaluate the potential of targeting CD38 as a plasma cell/plasmablast depletion mechanism by daratumumab in the treatment of patients with RA and SLE.MethodsRNA-sequencing analysis of synovial biopsies from various stages of RA disease progression, flow cytometry analysis of peripheral blood mononuclear cells (PBMC) from patients with RA or SLE and healthy donors, immunohistochemistry assessment (IHC) of synovial biopsies from patients with early RA, and ex vivo immune cell depletion assays using daratumumab (an anti-CD38 monoclonal antibody) were used to assess CD38 as a therapeutic target.ResultsWe demonstrated that the plasma cell/plasmablast-related genes CD38, XBP1, IRF4, PRDM1, IGJ and TNFSF13B are significantly up-regulated in synovial biopsies from patients with arthralgia, undifferentiated arthritis (UA), early RA and established RA as compared to healthy controls and control patients with osteoarthritis. In addition, the highest CD38 expression was observed on plasma cells and plasmablasts compared to natural killer (NK) cells, classical dendritic cells (DCs), plasmacytoid DCs (pDCs) and T cells, in blood from healthy controls and patients with SLE and RA. Furthermore, IHC showed CD38 staining in the same region as CD3 and CD138 staining in synovial tissue biopsies from patients with early RA. Most importantly, our data show for the first time that daratumumab effectively depletes plasma cells/plasmablasts in PBMC from patients with SLE and RA in a dose-dependent manner ex vivo.ConclusionThese results indicate that CD38 may be a potential target for RA disease interception and daratumumab should be evaluated clinically for the treatment of both RA and SLE.

Abstract C109: Quantitative analysis of multiple subtypes of immune system cells in cancer tissues

Current cancer biology acknowledges the key role of the immune system in tumor biology, and promise for the modulation of immune system in cancer treatment. The composition of the inflammatory cell populations in tissues is reflective of the overall state of the Tumor Micro-Environment (TME), and the identification of distinct inflammatory cell types may hold prognostic or predictive value. Immunohistochemistry allows for reliable identification of the cell constituents to facilitate analysis of the TME while remaining in the tissue context. Establishing a quantitative paradigm for inflammatory cell types and subtype profiling requires unbiased and automated whole-tissue based quantitation methods, which are capable of spatial integration of multiple inflammatory cell markers across the whole tissue. While single slide fluorescent multiplex approaches can address this need, the use of difficult-to-implement wet assay strategies involving multiplexing 6-8 fluorescent markers on the same tissue section are difficult to implement in a global clinical diagnostic lab setting. To answer this need, we combined novel advents in Tissue Image Analysis (TIA) to integrate spatial expression of serial-section stained whole tissue clinical lung cancer specimens. In this proof-of-principle study,we were able to superimpose specific locations of individual cell types onto 6 serial sections and evaluate different inflammatory cell types. We used serial sections of clinical lung specimens stained for six immune phenotypic markers (CD68, CD4, CD8, CD33, FoxP3, and CD11b) to illustrate a repertoire of inflammatory cell types. Our proprietary CellMap algorithm was utilized to identify, enumerate, and determine the precise location of individual inflammatory cells in tissues on cell-by-cell basis in the tumor microenvironment (TME). Our proprietary FACTS (Feature Analysis on Consecutive Tissue Sections) approach was used to integrate the spatial expression of individual markers onto a reference HE 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C109.

Quantitative paradigm for analysis of multiple subtypes of immune system cells in lung cancer tissues

The state of the immune system is reflected, in part, by the cell populations present in individual tissues which reflect the tumor microenvironment (TME). Several studies have suggested that understanding the TME constituents is useful for predicting drug response outcomes. Despite the biological significance of various inflammatory cell types in the TME, a widely accepted quantitative paradigm that allows the comparison of multiple subtypes of inflammatory cells in tissues is lacking. This is largely due to our inability to integrate key spatial information for multiple biomarkers across whole tissue sections, understand the concordance of individual biomarkers/cell types, account for heterogeneity in the region of analysis, and the absence of validated correlative scoring paradigms to measure against drug response. Finally, if these hurdles of understanding are met, a system needs to be created which can efficiently and reproducibly asses these readouts in a clinical-use environment. In this study, we revealed by immunohistochemistry distinct populations of immune system cells on serial sections of clinical lung cancer tissues. The immune cell markers evaluated included CD68, CD33, CD11b, FoxP3, CD4, and CD8. Whole tissue image analysis (tIA) was performed using Flagships CellMap™ algorithm tool for each biomarker. Biomarker expression was analyzed on cell by cell basis for the tumor microenvironment and tabulated across the whole tissues. In order to integrate spatial information of CD68, CD33, CD11b, FoxP3, CD4, and CD8 biomarker expression from each slide onto a single tissue section, we utilized Flagships patented FACTS™ tIA tool. This tool allows for an overlay of biomarker content information on to a single reference slide (H&E), and thus can provide key spatial information on biomarker expression relationships and presence of distinct cell populations in relation to each other. These studies directly demonstrate an effective utilization of combination of IHC with a set of Flagship tIA-based tools to score and analyze immune cell biomarker content in whole clinical tissue samples. Importantly, these tools can spatially correlate multiple biomarkers across serial sections of a single tissue. These approaches will lead to the development of standardized quantitative paradigms with predictive power in evaluating patient drug response.

Abstract 4666: Profiling mesothelin protein expression by immunohistochemistry and gene expression in adenocarcinoma and squamous cell carcinoma of lung

Mesothelin is a 40 kDa secreted glycoprotein expressed in normal mesothelial cells and over-expressed in several histological types of tumors. Detection of mesothelin by immunohistochemistry (IHC) may assist in the diagnosis of mesothelioma. Mesotheliomas are positive for mesothelin staining, but carcinomas of the lung may also be positive for this marker. Mesothelin positivity in adenocarcinoma of the lung reportedly ranges from 22% to 71% of cases depending upon the specific subtype. Mesothelin positivity in squamous cell carcinomas (SCC) of lung is reported to be 16% in non-keratinizing and 31% in keratinizing subtypes. We performed mesothelin IHC on a cohort of 50 lung carcinoma samples (16 adenocarcinomas and 34 SCC). Mesothelin expression was observed in 10 out of 16 (62.5%) lung adenocarcinoma samples, of which 8 showed greater than 10% of tumor cells with positive membrane staining of any intensity. Mesothelin expression was observed in 19 out of 34 (55.9%) lung SCC samples, of which only 3 samples showed greater than 10% of tumor cells with positive membrane staining of any intensity. These findings are in concordance with previous reports which show a higher prevalence of mesothelin protein expression in lung adenocarcinoma than in lung SCC. To correlate RNA expression with protein expression, we performed gene expression profiling on a subcohort of these lung cancer specimens. Out of 50 lung carcinoma samples, 28 samples (10 adenocarcinomas and 18 SCCs) provided adequate RNA yield for gene expression profiling for mesothelin. A total mean relative gene expression (mRGE) value of 13.64 with a standard deviation (SD) of ±3.71 was obtained for the adenocarcinoma samples and a mRGE value of 14.78 with a SD of 2.64 was obtained for the SCC samples. We next arbitrarily assigned samples with greater than 10% mesothelin stained cells as “positive” and the remainder as “negative”. Six negative adenocarcinoma samples yielded a mRGE value of 12.98 with a SD of ±3.84, and four positive adenocarcinoma samples yielded a mRGE value of 14.63 with SD of ±3.82. Sixteen negative SCC samples resulted in a mRGE 14.63 with SD of ± 2.75 and two positive SCC samples yielded a mRGE of 15.98 with a SD of ±1.37. There was no apparent correlation between mRGE values and IHC positivity. We also correlated gene expression with p53 mutation status. Sixteen wild type samples, composed of equal number of adenocarcinoma and SSC had a total mRGE of 14.29 with SD of ±3.32. Seven samples with p53 mutations had a total mRGE of 16.09 with SD ±1.93. These data indicate that mesothelin gene expression is not associated with p53 mutation status. Future studies with an increased number of samples may yield significant associations between protein expression, gene expression and mutation status. Citation Format: Jackson Wong, Dana Gaffney, Michael Sharp, Brenda Hertzog, Jayaprakash Karkera, Suso Platero, John Alvarez. Profiling mesothelin protein expression by immunohistochemistry and gene expression in adenocarcinoma and squamous cell carcinoma of lung. [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 4666. doi:10.1158/1538-7445.AM2014-4666

Abstract 1655: Human immune cell infiltration of tumors in a PBMC-humanized NSG mouse xenograft model changes with treatment of an anti-tissue factor antibody

Immunocompromised mice engrafted with human hematopoietic stem cells (HSC) or peripheral blood mononuclear cells (PBMC) can be promising models of human immune system compartments, useful for studying diseases such as diabetes, lupus, GVHD, and cancer. We have utilized the PBMC-humanized mouse as a platform for xenograft models. To investigate the effect of human antibody (Ab) treatment on immune cells and xenografts using this platform, we examined the infiltrate of human immune cells in A431 xenografts with treatment of an effector function-enhanced anti-tissue factor VIIA (TFVIIA) Ab. Female NSG mice were intravenously engrafted with human PBMC, and subcutaneously implanted two weeks later with A431 human SCC cells. One week post tumor cell implant and thereafter twice weekly, mice received treatment with anti-TFVIIA Ab. Peripheral blood, bone marrow and spleen were analyzed by FACS and terminal tumor samples were analyzed by FACS and immunohistochemistry (IHC). FACS analysis of blood from Ab treated and untreated tumor-bearing mice collected from baseline to termination, showed an overall increase in the frequency of human CD45+, CD3+, CD8+, and CD4+ cells, while the frequency of CD56+ cells remained stable. In comparison, non-tumor bearing mice had increased CD56+ cells and decreased CD3+ cells. FACS analysis of tumors showed a robust increase in the frequency of CD56+ cells and a decrease in the frequency of CD3+ cells with treatment of anti-TF Ab. With treatment, the frequency of CD8+ cells was markedly lower and the frequency of CD4+ cells unchanged. Histological analysis was performed on A431 FFPE xenografts from both the treated and non-treated groups. Hematoxylin and eosin (HE 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1655. doi:10.1158/1538-7445.AM2014-1655