Interim Analysis of Phase 2 Results for Cemiplimab in Patients with Metastatic Basal Cell Carcinoma (mBCC) who Progressed on or are Intolerant to Hedgehog Inhibitors (HHIs)

Abstract not available

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Circulating tumor cells in melanoma patients.

Circulating tumor cells (CTCs) are of recognized importance for diagnosis and prognosis of cancer patients. With melanoma, most studies do not show any clear relationship between CTC levels and stage of disease. Here, CTCs were enriched (∼400X) from blood of melanoma patients using a simple centrifugation device (OncoQuick), and 4 melanocyte target RNAs (TYR, MLANA, MITF, and MIF) were quantified using QPCR. Approximately one-third of melanoma patients had elevated MIF and MLANA transcripts (p<0.0001 and p<0.001, respectively) compared with healthy controls. In contrast, healthy controls had uniformly higher levels of TYR and MITF than melanoma patients (p<0.0001). There was a marked shift of leukocytes into the CTC-enriched fractions (a 430% increase in RNA recovery, p<0.001), and no relationship between CTC levels and stage of disease was found. CTCs were captured on microfabricated filters and cultured. Captured melanoma CTCs were large cells, and consisted of 2 subpopulations, based on immunoreactivity. One subpopulation (∼50%) stained for both pan-cytokeratin (KRT) markers and the common leukocyte marker CD-45, whereas the second subpopulation stained for only KRT. Since similar cells are described in many cancers, we also examined blood from colorectal and pancreatic cancer patients. We observed analogous results, with most captured CTCs staining for both CD-45/KRT markers (and for the monocyte differentiation marker CD-14). Our results suggest that immature melanocyte-related cells (expressing TYR and MITF RNA) may circulate in healthy controls, although they are not readily detectable without considerable enrichment. Further, as early-stage melanomas develop, immature melanocyte migration into the blood is somehow curtailed, whereas a significant proportion of patients develop elevated CTC levels (based on MIF and MLANA RNAs). The nature of the captured CTCs is consistent with literature describing leukocyte/macrophage-tumor cell fusion hybrids, and their role in metastatic progression

Macrophage-Tumor Cell Fusions from Peripheral Blood of Melanoma Patients

<div><p>Background</p><p>While the morbidity and mortality from cancer are largely attributable to its metastatic dissemination, the integral features of the cascade are not well understood. The widely accepted hypothesis is that the primary tumor microenvironment induces the epithelial-to-mesenchymal transition in cancer cells, facilitating their escape into the bloodstream, possibly accompanied by cancer stem cells. An alternative theory for metastasis involves fusion of macrophages with tumor cells (MTFs). Here we culture and characterize apparent MTFs from blood of melanoma patients.</p><p>Methods</p><p>We isolated enriched CTC populations from peripheral blood samples from melanoma patients, and cultured them. We interrogated these cultured cells for characteristic BRAF mutations, and used confocal microscopy for immunophenotyping, motility, DNA content and chromatin texture analyses, and then conducted xenograft studies using nude mice.</p><p>Findings</p><p>Morphologically, the cultured MTFs were generally large with many pseudopod extensions and lamellipodia. Ultrastructurally, the cultured MTFs appeared to be macrophages. They were rich in mitochondria and lysosomes, as well as apparent melanosomes. The cultured MTF populations were all heterogeneous with regard to DNA content, containing aneuploid and/or high-ploidy cells, and they typically showed large sheets (and/or clumps) of cytoplasmic chromatin. This cytoplasmic DNA was found within heterogeneously-sized autophagic vacuoles, which prominently contained chromatin and micronuclei. Cultured MTFs uniformly expressed pan-macrophage markers (CD14, CD68) and macrophage markers indicative of M2 polarization (CD163, CD204, CD206). They also expressed melanocyte-specific markers (ALCAM, MLANA), epithelial biomarkers (KRT, EpCAM), as well as the pro-carcinogenic cytokine MIF along with functionally related stem cell markers (CXCR4, CD44). MTF cultures from individual patients (5 of 8) contained melanoma-specific BRAF activating mutations. Chromatin texture analysis of deconvoluted images showed condensed DNA (DAPI-intense) regions similar to focal regions described in stem cell fusions. MTFs were readily apparent in vivo in all human melanomas examined, often exhibiting even higher DNA content than the cultured MTFs. When cultured MTFs were transplanted subcutaneously in nude mice, they disseminated and produced metastatic lesions at distant sites.</p><p>Conclusions and Hypothesis</p><p>Apparent MTFs are present in peripheral blood of patients with cutaneous melanomas, and they possess the ability to form metastatic lesions when transplanted into mice. We hypothesize that these MTFs arise at the periphery of primary tumors in vivo, that they readily enter the bloodstream and invade distant tissues, secreting cytokines (such as MIF) to prepare “niches” for colonization by metastasis initiating cells.</p></div

Metastatic Foci after subcutaneous implantation of Cultured MTFs in Athymic Nude Mice.

<p>MTFs from 2 separate patient samples were grown in culture for ~ 4 weeks, and 5 x 10⁵ cells were subcutaneously implanted in hind limbs of nude mice. Mice were sacrificed and necropsied 47 days later. Sections were taken from multiple locations and examined for human MTFs. Shown are two distinct metastatic foci in mouse pancreas, stained with antibodies specific for human CD204 (Upper row), MLANA (Middle row), and CD206 (Lower row). Left column shows a low power (20X) view, and Right column shows a higher power (40X) view of 2 distinct foci. Inset in the Left Middle Panel shows a blown-up view of a single binucleate human cell in mouse pancreas, stained for human MLANA. Many of the cells in the foci also contained pigment (melanin), which was visible on standard H&E-stained sections.</p

Representative confocal images of MTFs in primary human melanomas.

<p>Cells were stained for nuclei with DAPI (Blue), shown in Panels [<b>A</b>, <b>E</b>, <b>I</b>, <b>M</b>, <b>Q</b>, <b>U</b>, and <b>Y</b>]. The same cells were also stained with various fluorescent markers specific for melanocyte, macrophage, or epithelial differentiation, and images are shown in rows. Panels <b>[B,C]</b>: Melanocyte marker MLANA (Red) and M2- polarization macrophage marker CD204 (Green). Panels <b>[F,G]</b>: Melanocyte marker MLANA (Red) and M2- polarization macrophage marker CD206 (Green). Panels <b>[J,K]</b>: Melanocyte marker MLANA (Red) and M2- polarization macrophage marker CD163 (Green). Panels <b>[N,O]</b>: Melanocytic marker ALCAM (Green) and M2- polarization macrophage marker CD206 (Red). Panels <b>[R,S]</b>: M2- polarization Macrophage marker CD206 (Red) and epithelial marker pKRT (Green). Panels <b>[V,W,Z, and A1]</b>: M2- polarization Macrophage marker CD163 (Green) and epithelial cell adhesion molecule EpCAM (Red). As is evident, there are distinct populations of cells (in each of 6 primary melanoma specimens examined, as well as the metastatic lesions) which dual-stain for macrophage-melanocyte markers, which are often seen surrounding nests of melanoma cells. These cells also stain for epithelial markers. Composite images are shown in Panels [<b>D</b>, <b>H</b>, <b>L</b>, <b>P</b>, <b>T</b>, <b>X</b>, and <b>B1</b>]. Panels underneath Panels [<b>A-D</b>] represent XZ views of the panels above.</p

Immunostaining of Cultured MTFs.

<p>Representative confocal images of cultured MTFs. Nuclei were stained with DAPI (Blue); individual cells are shown in Panels [<b>A</b>,<b>E</b>,<b>I</b>,<b>M</b>, and <b>Q</b>]). The same cells were also stained with pairs of various fluorescent markers specific for melanocyte, macrophage, or epithelial differentiation (shown in each row). Panel <b>[B,C]</b>: Melanocytic marker ALCAM (Green) and M2- polarization macrophage marker CD204 (Red). Panel <b>[F,G]</b>: Pan-Cytokeratin (Green) and M2- polarization macrophage marker CD204 (Red). Panel <b>[J,K]</b>: M2- polarization Macrophage marker CD206 (Green) and epithelial cell adhesion molecule EpCAM (Red). Panel <b>[N,O]</b>: Melanocyte marker MLANA (Green) and M2- polarization macrophage marker CD204 (Red). Panel <b>[R,S]</b>: Melanocyte marker MLANA (Green) and M2- polarization macrophage marker CD206 (Red). Composite images are shown in Panels [<b>D</b>, <b>H</b>, <b>L</b>, <b>P</b>, and <b>T</b>].</p