Combination anti-CXCR4 and anti-PD-1 immunotherapy provides survival benefit in glioblastoma through immune cell modulation of tumor microenvironment.

BACKGROUND: Emerging evidence suggests that myeloid cells play a critical role in glioblastoma (GBM) immunosuppression. Disappointing results of recent checkpoint inhibitor trials suggest that combination immunotherapy with alternative agents could be fruitful in overcoming immunosuppression. Overexpression of chemokine receptor CXCR4 is associated with poor prognosis in GBM. We investigate the treatment effects of combination immunotherapy with anti-PD-1 and anti-CXCR4 in a murine glioma model. METHODS: C57BL/6 mice were implanted with GL261-Luc+ glioma cells and randomized into 4 arms: (1) control (2) anti-PD-1 (3) anti-CXCR4, and (4) anti-PD-1 and anti-CXCR4 therapy. Overall survival and median survival were assessed. Cell populations were assessed by flow cytometry. RESULTS: Combination therapy conferred a significant survival benefit compared to control and monotherapy arms. Mice that received combination therapy demonstrated immune memory and decreased populations of immunosuppressive tumor-infiltrating leukocytes, such as monocytic myeloid-derived suppressor cells and microglia within the brain. Furthermore, combination therapy improved CD4+/CD8+ ratios in the brain as well as contributed to increased levels of pro-inflammatory cytokines. CONCLUSIONS: Anti-CXCR4 and anti-PD-1 combination immunotherapy modulates tumor-infiltrating populations of the glioma microenvironment. Targeting myeloid cells with anti-CXCR4 facilitates anti-PD-1 to promote an antitumor immune response and improved survival rates.

Assessment of the Impact of Alternative Fixatives on HER2 Detection in Breast Cancer and Gastric Cancer Tumor Specimens.

The type of fixative used for preserving tumor specimens can significantly impact the performance of the immunohistochemistry and in situ hybridization assays used for assessing human epidermal growth factor receptor 2 (HER2) status. This study reports the prevalence of the use of alternative fixatives other than the guideline-recommended 10% neutral buffered formalin (NBF) during HER2 testing in a real-world setting. The effects of alternative fixatives [20% NBF and 10% unbuffered formalin (UBF) fixatives] on HER2 testing of breast cancer (BC) and gastric cancer (GC) cell lines and tissues are also assessed. Overall, 117,636 tumor samples received at a central laboratory from >8000 clinical trial sites across 60 countries were reviewed to determine the prevalence of alternative fixative usage. To investigate the impact of alternative fixatives, 27 cell lines (21 BC and 6 GC) and 76 tumor tissue samples (50 BC and 26 GC) were fixed in 10% NBF, 20% NBF, or 10% UBF, and evaluated for HER2 status by immunohistochemistry and in situ hybridization. Real-world data showed that 9195 (7.8%) tumor samples were preserved using an alternative fixative. In cell lines, overall percentage agreement, negative percentage agreement, and positive percentage agreement among the 3 fixatives were 100%. In tumor tissues, the agreement among 10% NBF, 20% NBF, and 10% UBF ranged between 94.7% and 96.6% for negative percentage agreement and 90.9% for overall percentage agreement compared with a range of 58.3% to 66.7% for positive percentage agreement. These results suggest that alternative fixatives may have the potential to convert HER2 status in tissues from positive to negative.

An immune-active tumor microenvironment favors clinical response to ipilimumab.

PURPOSE: Ipilimumab, a fully human monoclonal antibody specific to CTLA-4, has been shown to improve overall survival in metastatic melanoma patients. As a consequence of CTLA-4 blockade, ipilimumab treatment is associated with proliferation and activation of peripheral T cells. To better understand various tumor-associated components that may influence the clinical outcome of ipilimumab treatment, gene expression profiles of tumors from patients treated with ipilimumab were characterized. EXPERIMENTAL DESIGN: Gene expression profiling was performed on tumor biopsies collected from 45 melanoma patients before and 3 weeks after the start of treatment in a phase II clinical trial. RESULTS: Analysis of pre-treatment tumors indicated that patients with high baseline expression levels of immune-related genes were more likely to respond favorably to ipilimumab. Furthermore, ipilimumab appeared to induce two major changes in tumors from patients who exhibited clinical activity: genes involved in immune response showed increased expression, whereas expression of genes for melanoma-specific antigens and genes involved in cell proliferation decreased. These changes were associated with the total lymphocyte infiltrate in tumors, and there was a suggestion of association with prolonged overall survival in these patients. Many IFN-γ-inducible genes and Th1-associated markers showed increased expression after ipilimumab treatment, suggesting an accumulation of this particular type of T cell at the tumor sites, which might play an important role in mediating the antitumor activity of ipilimumab. CONCLUSIONS: These results support the proposed mechanism of action of ipilimumab, suggesting that cell-mediated immune responses play an important role in the antitumor activity of ipilimumab.

Reliable Gene Expression Profiling from Small and Hematoxylin and Eosin-Stained Clinical Formalin-Fixed, Paraffin-Embedded Specimens Using the HTG EdgeSeq Platform.

Clinical biomarker studies are often hindered by the scarcity or suboptimal quality of biological specimens. EdgeSeq, a transcriptomics analysis platform, combines quantitative nuclease protection assay technology with next-generation sequencing, using small amounts of starting material and delivering reproducible gene expression profiles from challenging material, such as formalin-fixed, paraffin-embedded (FFPE) tissue. To evaluate EdgeSeq for analysis of archives of stained FFPE tissue, EdgeSeq was performed on unstained, hematoxylin and eosin (H&E)-stained, and immunohistochemistry-stained slides from patients with small-cell and non-small-cell lung cancer. Pairwise comparisons of gene expression profiles from stained and unstained slides showed higher Pearson correlation coefficients with H&E staining (0.86 to 0.97) than with immunohistochemistry staining (0.21 to 0.56). A 25-gene interferon-γ signature score from unstained slides showed a Pearson correlation coefficient of 0.92 with H&E-stained slides and a significant Spearman correlation (P = 0.0025) with immune scores. To test gene expression profiling in small samples, FFPE sample equivalents were examined from 5.0 to 0.08 mm2 of a section (5 µm thick); sample equivalents ≥0.31 mm2 showed alignment rates >69% and pairwise Pearson correlation coefficients ≥0.87. EdgeSeq can, thus, be used to profile small and H&E-stained FFPE tumor specimens to obtain biomarker data from limited tissue in oncology clinical trials and enable research into tumor microenvironment and immune cell engagement with tumors at the locoregional level.

Identification of miRNA changes in Alzheimer's disease brain and CSF yields putative biomarkers and insights into disease pathways.

MicroRNAs have essential functional roles in brain development and neuronal specification but their roles in neurodegenerative diseases such as Alzheimer's disease (AD) is unknown. Using a sensitive qRT-PCR platform we identified regional and stage-specific deregulation of miRNA expression in AD patient brains. We used experimental validation in addition to literature to reveal how the deregulated brain microRNAs are biomarkers for known and novel pathways in AD pathogenesis related to amyloid processing, neurogenesis, insulin resistance, and innate immunity. We additionally recovered miRNAs from cerebrospinal fluid and discovered AD-specific miRNA changes consistent with their role as potential biomarkers of disease.

Development of a Diagnostic Programmed Cell Death 1-Ligand 1 Immunohistochemistry Assay for Nivolumab Therapy in Melanoma.

Nivolumab is a monoclonal antibody that blocks the interaction between programmed cell death 1 (PD1) and programmed cell death 1-ligand 1 (PD-L1), resulting in enhanced antitumor activity by the immune system. Nivolumab is currently approved by the US Food and Drug Administration (FDA) for melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma, classical Hodgkin lymphoma, squamous cell carcinoma of the head and neck, and urothelial carcinoma. PD-L1 IHC 28-8 pharmDx is FDA-approved as a complementary diagnostic for immunohistochemical (IHC) detection of PD-L1 in non-squamous NSCLC and melanoma. We report validation of PD-L1 IHC 28-8 pharmDx for PD-L1 detection on formalin-fixed, paraffin-embedded human melanoma specimens using Autostainer Link 48. A prevalence assessment of 104 melanoma specimens indicated that PD-L1 was detected across the full expression level range (0% to 100% of tumor cells). Assay robustness and precision studies were conducted at Agilent Technologies, with additional reproducibility studies performed at 3 external laboratories. Precision studies evaluated at ≥1% and ≥5% expression levels revealed a range of average negative agreement from 89.5%, 95% CI (83.2, 93.6) to 100%, 95% CI (97.3, 100), and average positive agreement from 85.5%, 95% CI (77.6, 90.9) to 100%, 95% CI (97.9, 100). For external reproducibility, precise results were obtained. These results demonstrate PD-L1 IHC 28-8 pharmDx is a precise, robust, and reproducible assay for determining PD-L1 expression in melanoma. This is the first PD-L1 IHC test to receive FDA approval as a complementary diagnostic in melanoma patients whereby positive PD-L1 expression is correlated with the magnitude of nivolumab treatment effect.

An Analytical Comparison of Dako 28-8 PharmDx Assay and an E1L3N Laboratory-Developed Test in the Immunohistochemical Detection of Programmed Death-Ligand 1.

AIM: Nivolumab, a fully human immunoglobulin G4 programmed death-1 (PD-1) immune checkpoint inhibitor antibody, has activity in melanoma, non-small-cell lung cancer (NSCLC), renal cell carcinoma (RCC), and Hodgkin lymphoma. Nivolumab is approved in the USA and EU for advanced melanoma, NSCLC, and RCC, and relapsed Hodgkin lymphoma in the USA. Programmed death-ligand 1 (PD-L1), a PD-1 ligand, is expressed on mononuclear leukocytes, myeloid cells, and tumor cells. PD-L1 is being investigated as a potential biomarker to predict the association of tumor PD-L1 expression with nivolumab efficacy. METHODS: Bristol-Myers Squibb and Dako previously reported on an automated PD-L1 immunohistochemical (IHC) assay that detects cell surface PD-L1 in formalin-fixed, paraffin-embedded, human tumor tissue specimens using Dako's Autostainer Link 48. The primary antibody for this assay is a rabbit monoclonal antihuman PD-L1 antibody, clone 28-8. Another rabbit monoclonal antihuman PD-L1 antibody, clone E1L3N, was compared with 28-8 for specificity and sensitivity using an identical detection method followed by vendor-recommended detection methods. RESULTS: Using PD-L1 null clones of L2987 and ES-2 tumor cell lines, both antibodies were specific for detection of PD-L1 on the plasma membrane, although E1L3N also stained cytoplasm in ES-2 knockout cells. Using the identical method, E1L3N was slightly more sensitive than 28-8 based on staining intensities. Using manufacturer-recommended detection methods and predefined scoring criteria for plasma membrane staining of tumor and immune cells, 28-8 demonstrated significantly improved detection compared with E1L3N. CONCLUSIONS: Epitope retrieval and highly sensitive detection reagents are key determinants in IHC detection of PD-L1.

Evaluation and selection of a non-PCR based technology for improved gene expression profiling from clinical formalin-fixed, paraffin-embedded samples.

AIM: Formalin-fixed, paraffin-embedded (FFPE) clinical tissue samples have the potential to provide valuable gene-expression data for the development of cancer biomarkers. However, FFPE RNA is extensively fragmented, presenting a significant challenge for reliably detecting gene expression using traditional qPCR methods. RESULTS: We evaluated three novel methodologies along with the traditional qPCR method for their ability to detect Notch pathway gene expression in colorectal cancer FFPE tissue RNAs. We found that quantitative nuclease protection assay-detected gene expression in high-quality RNAs as sensitively as qPCR, and consistently detected mRNAs in highly fragmented FFPE tissue RNAs. CONCLUSION: Quantitative nuclease protection assay represents an improved methodology for detecting gene expression in FFPE tissue and has the potential to advance the development of cancer biomarkers.

Genotoxic stress-induced activation of Plk3 is partly mediated by Chk2.

Polo-like kinase 3 (Plk3, alternatively termed Prk) is involved in the regulation of DNA damage checkpoint as well as in M-phase function. Plk3 physically interacts with p53 and phosphorylates this tumor suppressor protein on serine-20, suggesting that the role of Plk3 in cell cycle progression is mediated, at least in part, through direct regulation of p53. Here we show that Plk3 is rapidly activated by reactive oxygen species in normal diploid fibroblast cells (WI-38), correlating with a subsequent increase in p53 protein level. Plk3 physically interacts with Chk2 and the interaction is enhanced upon DNA damage. In addition, Chk2 immunoprecipitated from cell lysates of Daudi (which expressed little Plk3) is capable of stimulating the kinase activity of purified recombinant Plk3 in vitro, and this stimulation is more pronounced when Plk3 is supplemented with Chk2 immunoprecipitated from Daudi after DNA damage. Furthermore, ectopic expression Chk2 activates cellular Plk3. Together, our studies suggest Chk2 may mediate direct activation of Plk3 in response to genotoxic stresses.

Development of an automated PD-L1 immunohistochemistry (IHC) assay for non-small cell lung cancer.

Nivolumab, a fully human IgG4 programmed death 1 (PD-1) immune checkpoint inhibitor antibody, developed by Bristol-Myers Squibb Inc., has activity across non-small cell lung cancer (NSCLC) histologies and is Food and Drug Administration approved for treatment of metastatic squamous NSCLC with progression on or after platinum-based chemotherapy. PD-L1 has been investigated as a potential biomarker to predict clinical response to nivolumab in clinical settings. We report an automated PD-L1 immunohistochemistry (IHC) assay, which was developed to detect cell surface PD-L1 in formalin-fixed paraffin-embedded human tumor tissue specimens using Dako's Autostainer Link 48. The primary antibody for this assay is a rabbit monoclonal anti-human PD-L1 antibody, clone 28-8. The specificity of 28-8 for PD-L1 was demonstrated by antigen competition and genetic deletion of PD-L1 in tumor cell lines. The specificity of the PD-L1 IHC assay was further evaluated in a collection of 30 normal human tissues. The PD-L1 IHC assay was optimized for high sensitivity and precision in routine application. A pathology scoring and interpretation method specific to nivolumab clinical studies was adopted for the assay. The analytical performance of the assay was validated for application in the determination of PD-L1 status in human NSCLC specimens. The clinical application of the assay and scoring method was further validated in 3 Clinical Laboratory Improvement Amendments certified labs. The assay is currently being investigated in a variety of clinical studies for use as an in vitro diagnostic to select and stratify patients for treatment with the anti-PD-1 therapeutic antibody, nivolumab.

IRS2 copy number gain, KRAS and BRAF mutation status as predictive biomarkers for response to the IGF-1R/IR inhibitor BMS-754807 in colorectal cancer cell lines.

Insulin-like growth factor receptor 1 (IGF-1R)-targeting therapies are currently at an important crossroad given the low clinical response rates seen in unselected patients. Predictive biomarkers for patient selection are critical for improving clinical benefit. Coupling in vitro sensitivity testing of BMS-754807, a dual IGF-1R/IR inhibitor, with genomic interrogations in 60 human colorectal cancer cell lines, we identified biomarkers correlated with response to BMS-754807. The results showed that cell lines with BRAF(V600E) or KRAS(G13D) mutation were resistant, whereas cell lines with wild-type of both KRAS and BRAF were particularly sensitive to BMS-754807 if they have either higher RNA expression levels of IR-A or lower levels of IGFBP6. In addition, the cell lines with KRAS mutations, those with either insulin receptor substrate 2 (IRS2) copy number gain (CNG) or higher IGF-1R expression levels, were more sensitive to the drug. Furthermore, cell lines with IRS2 CNG had higher levels of ligand-stimulated activation of IGF-1R and AKT, suggesting that these cell lines with IGF-IR signaling pathways more actively coupled to AKT signaling are more responsive to IGF-1R/IR inhibition. IRS2 siRNA knockdown reduced IRS2 protein expression levels and decreased sensitivity to BMS-754807, providing evidence for the functional involvement of IRS2 in mediating the drug response. The prevalence of IRS2 CNG in colorectal cancer tumors as measured by qPCR-CNV is approximately 35%. In summary, we identified IRS2 CNG, IGF-1R, IR-A, and IGFBP6 RNA expression levels, and KRAS and BRAF mutational status as candidate predictive biomarkers for response to BMS-754807. This work proposed clinical development opportunities for BMS-754807 in colorectal cancer with patient selection to improve clinical benefit.

Mutations in the hedgehog pathway genes SMO and PTCH1 in human gastric tumors.

The causal role of the hedgehog pathway in cancer has been best documented in basal cell carcinoma of the skin. To assess potential DNA alterations of the hedgehog pathway in gastric cancer, we sequenced SMO and PTCH1 genes in a set of 39 gastric tumors. Tumors were classified by histology based on the Lauren classification and Sanger sequencing was performed to obtain full length coding sequences. Genomic instability was evident in these tumors as a number of silent or missense mutations were found. In addition to those that are potential germline polymorphisms, we found three SMO missense mutations, and one PTCH1 frameshift mutation that are novel and have not been documented in basal cell carcinoma. Mutations were found in both intestinal and diffuse type gastric tumors as well as in tumors that exhibit both intestinal and diffuse features. mRNA expression of hedgehog pathway genes was also examined and their levels do not indicate unequivocal higher pathway activity in tumors with mutations than those without. In summary, SMO and/or PTCH1 mutations are present at low frequency in different histologic subtypes of gastric tumors and these do not appear to be driver mutations.

Polo-like kinases and the microtubule organization center: targets for cancer therapies.

Studies from eukaryotic model systems, ranging from yeast to human, indicate that Polo and Polo-like kinases (Plks) are essential for the activity of the microtubule organization center. Polo/Plks localize to centrosomes or spindle pole bodies and undergo dramatic subcellular relocation during the cell cycle. Deregulated activities of Plks often result in abnormalities in centrosome duplication, maturation, and/or microtubule dynamics. Genetic and biochemical approaches have identified several candidate genes that either lie in the same pathway as POLO/PLKs or whose products are direct targets of Polo/Plks during the centrosome cycle. Recent studies have demonstrated that mammalian Plks also regulate the function of the Golgi complex, a cellular organelle closely associated with the centrosome and also having microtubule organization activity. Furthermore, deregulated expression of human PLK1 and PLK3 is strongly correlated with the development of many types of malignancies, and ectopic expression of kinase-active Plk3 or Plk1 dominant negative protein leads to rapid cell death. Given that several effective anti-tumor drugs directly interfere with microtubule dynamics, mammalian Plks are excellent targets for the development of anticancer drugs.