Avelumab, an anti-PD-L1 antibody, in patients with locally advanced or metastatic breast cancer: a phase 1b JAVELIN Solid Tumor study

PURPOSE: Agents targeting programmed death receptor 1 (PD-1) or its ligand (PD-L1) have shown antitumor activity in the treatment of metastatic breast cancer (MBC). The aim of this study was to assess the activity of avelumab, a PD-L1 inhibitor, in patients with MBC. METHODS: In a phase 1 trial (JAVELIN Solid Tumor; NCT01772004), patients with MBC refractory to or progressing after standard-of-care therapy received avelumab intravenously 10 mg/kg every 2 weeks. Tumors were assessed every 6 weeks by RECIST v1.1. Adverse events (AEs) were graded by NCI-CTCAE v4.0. Membrane PD-L1 expression was assessed by immunohistochemistry (Dako PD-L1 IHC 73-10 pharmDx). RESULTS: A total of 168 patients with MBC, including 58 patients with triple-negative breast cancer (TNBC), were treated with avelumab for 2-50 weeks and followed for 6-15 months. Patients were heavily pretreated with a median of three prior therapies for metastatic or locally advanced disease. Grade >/= 3 treatment-related AEs occurred in 13.7% of patients, including two treatment-related deaths. The confirmed objective response rate (ORR) was 3.0% overall (one complete response and four partial responses) and 5.2% in patients with TNBC. A trend toward a higher ORR was seen in patients with PD-L1+ versus PD-L1- tumor-associated immune cells in the overall population (16.7% vs. 1.6%) and in the TNBC subgroup (22.2% vs. 2.6%). CONCLUSION: Avelumab showed an acceptable safety profile and clinical activity in a subset of patients with MBC. PD-L1 expression in tumor-associated immune cells may be associated with a higher probability of clinical response to avelumab in MBC

Matched rabbit monoclonal antibodies against αv-series integrins reveal a novel αvβ3-LIBS epitope, and permit routine staining of archival paraffin samples of human tumors

Summary The relationship between integrin expression and function in pathologies is often contentious as comparisons between human pathological expression and expression in cell lines is difficult. In addition, the expression of even integrins αvβ6 and αvβ8 in tumor cell lines is not comprehensively documented. Here, we describe rabbit monoclonal antibodies (RabMabs) against the extracellular domains of αv integrins that react with both native integrins and formalin fixed, paraffin embedded (FFPE) human tissues. These RabMabs, against αvβ3 (EM22703), αvβ5 (EM09902), αvβ6 (EM05201), αvβ8 (EM13309), and pan-αv (EM01309), recognize individual integrin chains in Western blots and in flow cytometry. EM22703 detected a ligand-induced binding site (LIBS), reporting an epitope enhanced by the binding of an RGD-peptide to αvβ3. αvβ8 was rarely expressed in human tumor specimens, and weakly expressed in non-small-cell lung carcinoma (NSCLC). However, ovarian carcinoma cell lines expressed αvβ8, as did some melanoma cells, whereas U87MG glioma lacked αvβ8 expression. We observed an unexpected strong expression of αvβ6 in tumor samples of invasive ductal breast adenoma, colorectal carcinoma (CRC), and NSCLC. αvβ3 was strongly expressed in some invasive NSCLC cohorts. Interestingly, PC3 prostate cell and human prostate tumors did not express αvβ3. The RabMabs stained plasma membranes in FFPE-immunohistochemistry (IHC) samples of tumor cell lines from lung, ovary, colon, prostate, squamous cell carcinoma of head and neck (SCCHN), breast, and pancreas carcinomas. The RabMabs are unique tools for probing αv integrin biology, and suggest that especially αvβ6 and αvβ8 biologies still have much to reveal

PD-L1 immunostaining scoring for non-small cell lung cancer based on immunosurveillance parameters - Table 2

<p>PD-L1 immunostaining scoring for non-small cell lung cancer based on immunosurveillance parameters</p> - Table

Relationships between biomarkers in our NSCLC cohort.

<p><b>(A)</b> Hierarchical clustering heat map of percentages of positive cells for FOXP3, PD-L1, CD8, Granzyme B, and CD68. Each biomarker (row) was normalized before clustering to give a mean of 0 and a standard deviation of 1 across columns (samples). The color scale indicates the relative size of the biomarker score compared to the other samples in the cohort with blue = low, white = neutral, and brown = high. Bracket height indicates the degree of correlation between measurements with shorter brackets representing higher correlations and taller brackets representing smaller correlations. <b>(B)</b> Heatmap of Pearson correlation coefficients between percent positive cells measurements across samples for FOXP3, PD-L1, CD8, Granzyme B, and CD68. The color scale ranges from the minimum (blue) and maximum (red) correlation coefficient observed in this analysis. Therefore red represents strong positive correlations, white represents moderate correlations, and blue represents weak correlations. No strong negative correlations were observed.</p

Examples of PD-L1 staining patterns observed and categorized in this NSCLC series.

<p><b>A</b>: Pattern I—Constitutive mixed with induced. Diffuse expression of PD-L1 (IHC) on tumor cell membranes of a squamous cell carcinoma, including central regions of trabeculae. Prominent labeling of cells in the TME compartment at the tumor-nest-TME interface suggesting presence of an immunological synapse (inset arrow). <b>B</b>: Pattern II—Induced only. Patchy expression of PD-L1 in a squamous cell carcinoma at the tumor-nest-TME interface (inset arrow). Minimal to no PD-L1 expression in the trabeculae (asterisk) if compared with (<b>A</b>). <b>C</b>: Pattern III—Immune ignorance. No to minimal PD-L1 expression in both tumor and TME compartments in an adenocarcinoma. <b>D</b>: Pattern IV—Constitutive only. Diffuse expression of PD-L1 by tumor-nests in an adenocarcinoma with minimal TME staining. <b>E</b>: Pattern V—TME expression only. No to minimal PD-L1 expression in tumor cells of a squamous cell carcinoma, with widespread staining in the TME compartment. <b>F</b>: Percentages of PD-L1-positive tumor cells in all staining pattern categories (I-V). <b>G</b>: Percentages of TME PD-L1-positive cells in all staining pattern categories (I-V). <b>H</b>: Digital H-scores from tumor-nests in all staining pattern categories (I-V). Significant differences between groups I and II were calculated using two-tailed Student’s t-tests, assuming unequal sample variances (*<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.01, ***<i>P</i> ≤ 0.001 and ****<i>P</i> ≤ 0.0001). Scale bars: 200 μm (<b>A-E</b>). NSCLC = non-small cell lung carcinoma; IHC = immunohistochemistry; TME = tumor microenvironment.</p

Classification of NSCLC according to PD-L1 expression patterns in tumor-nest and TME compartments.

<p>Classification of NSCLC according to PD-L1 expression patterns in tumor-nest and TME compartments.</p

Immune markers in NSCLC relative to a 50% cut-off point for PD-L1 staining.

<p><b>A</b>: Percentage of positive cells for each biomarker in “All Cells” (tumor nest and TME compartments combined) calculated by using Computational Tissue Analysis (cTA^™). NSCLC with >50% PD-L1-positive tumor cells were compared with those with lower expression. Statistical differences were calculated using two-tailed Student’s t-tests, assuming unequal sample variances (*<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.01, ***<i>P</i> ≤ 0.001 and ****<i>P</i> ≤ 0.0001). Percentages of cells positive for CD68, CD8, FoxP3, and Granzyme B were significantly higher in the >50% PD-L1 group. <b>B</b>: Representative IHC images of PD-L1-, CD8-, and CD68-positive cells (the most numerous biomarkers) in an NSCLC (solid carcinoma) case with >50% PD-L1 expression in the tumor nest compartment, and an NSCLC (adenocarcinoma with mixed subtypes) case with lower PD-L1 expression. Scale bars = 100 μm. NSCLC = non-small cell lung carcinoma; IHC = immunohistochemistry; TME = tumor microenvironment.</p

Example of computational tissue analysis (cTA) of NSCLC sections.

<p><b>A</b>: Manual inclusion (green line) and exclusion (red line) annotations in squamous cell carcinoma tissue for CD56 IHC. <b>B</b>: Enlarged view area from (<b>A)</b> showing exclusion annotation around a central necrotic area in tumor-nest (arrow) and a segment of the inclusion annotation around the tumor mass margin (arrowhead). <b>C</b>: PD-L1 IHC of a squamous cell carcinoma. <b>D</b>: Algorithm markup from (<b>C)</b> showing membrane scoring only in cells assigned to the tumor-nest compartment, with 1+ = yellow, 2+ = orange, and 3+ = red. The separated TME compartment cell nuclei are marked in green. Scale bars: 1 mm (<b>A</b>); 100 μm (<b>B</b>); and 150 μm (<b>C</b> and <b>D</b>). NSCLC = non-small cell lung carcinoma; IHC = immunohistochemistry; TME = tumor microenvironment.</p

Patient’s clinical information.

<p>Patient’s clinical information.</p