Phase II Clinical Trial of Pembrolizumab and Chemotherapy Reveals Distinct Transcriptomic Profiles by Radiologic Response in Metastatic Triple-Negative Breast Cancer.

PURPOSE: A single arm, phase II trial of carboplatin, nab-paclitaxel, and pembrolizumab (CNP) in metastatic triple-negative breast cancer (mTNBC) was designed to evaluate overall response rate (ORR), progression-free survival (PFS), duration of response (DOR), safety/tolerability, overall survival (OS), and identify pathologic and transcriptomic correlates of response to therapy. PATIENTS AND METHODS: Patients with ≤2 prior therapies for metastatic disease were treated with CNP regardless of tumor programmed cell death-ligand 1 status. Core tissue biopsies were obtained prior to treatment initiation. ORR was assessed using a binomial distribution. Survival was analyzed via the Kaplan-Meier method. Bulk RNA sequencing was employed for correlative studies. RESULTS: Thirty patients were enrolled. The ORR was 48.0%: 2 (7%) complete responses (CR), 11 (41%) partial responses (PR), and 8 (30%) stable disease (SD). The median DOR for patients with CR or PR was 6.4 months [95% confidence interval (CI), 4-8.5 months]. For patients with CR, DOR was >24 months. Overall median PFS and OS were 5.8 (95% CI, 4.7-8.5 months) and 13.4 months (8.9-17.3 months), respectively. We identified unique transcriptomic landscapes associated with each RECIST category of radiographic treatment response. In CR and durable PR, IGHG1 expression was enriched. IGHG1high tumors were associated with improved OS (P = 0.045) and were concurrently enriched with B cells and follicular helper T cells, indicating IGHG1 as a promising marker for lymphocytic infiltration and robust response to chemo-immunotherapy. CONCLUSIONS: Pretreatment tissue sampling in mTNBC treated with CNP reveals transcriptomic signatures that may predict radiographic responses to chemo-immunotherapy.

Immunotherapy with IL12 and PD1/CTLA4 inhibition is effective in advanced ovarian cancer and associates with reversal of myeloid cell-induced immunosuppression.

The tumor microenvironment (TME) in ovarian cancer (OC) is characterized by immune suppression, due to an abundance of suppressive immune cells populations. To effectively enhance the activity of immune checkpoint inhibition (ICI), there is a need to identify agents that target these immunosuppressive networks while promoting the recruitment of effector T cells into the TME. To this end, we sought to investigate the effect of the immunomodulatory cytokine IL12 alone or in combination with dual-ICI (anti-PD1 + anti-CTLA4) on anti-tumor activity and survival, using the immunocompetent ID8-VEGF murine OC model. Detailed immunophenotyping of peripheral blood, ascites, and tumors revealed that durable treatment responses were associated with reversal of myeloid cell-induced immune suppression, which resulted in enhanced anti-tumor activity by T cells. Single cell transcriptomic analysis further demonstrated striking differences in the phenotype of myeloid cells from mice treated with IL12 in combination with dual-ICI. We also identified marked differences in treated mice that were in remission compared to those whose tumors progressed, further confirming a pivotal role for the modulation of myeloid cell function to allow for response to immunotherapy. These findings provide the scientific basis for the combination of IL12 and ICI to improve clinical response in OC.

Mediators of Inflammation-Driven Expansion, Trafficking, and Function of Tumor-Infiltrating MDSCs.

Myeloid-derived suppressor cells (MDSC) are induced by and accumulate within many histologically distinct solid tumors, where they promote disease by secreting angiogenic and immunosuppressive molecules. Although IL1ß can drive the generation, accumulation, and functional capacity of MDSCs, the specific IL1ß-induced inflammatory mediators contributing to these activities remain incompletely defined. Here, we identified IL1ß-induced molecules that expand, mobilize, and modulate the accumulation and angiogenic and immunosuppressive potencies of polymorphonuclear (PMN)-MDSCs. Unlike parental CT26 tumors, which recruited primarily monocytic (M)-MDSCs by constitutively expressing GM-CSF- and CCR2-directed chemokines, IL1ß-transfected CT26 produced higher G-CSF, multiple CXC chemokines, and vascular adhesion molecules required for mediating infiltration of PMN-MDSCs with increased angiogenic and immunosuppressive properties. Conversely, CT26 tumors transfected with IL1ß-inducible molecules could mobilize PMN-MDSCs, but because they lacked the ability to upregulate IL1ß-inducible CXCR2-directed chemokines or vascular adhesion molecules, additional PMN-MDSCs could not infiltrate tumors. IL1ß-expressing CT26 increased angiogenic and immunosuppressive factors of tumor-infiltrating MDSCs, as did CT26 tumors individually transfected with G-CSF, Bv8, CXCL1, or CXCL5, demonstrating that mediators downstream of IL1ß could also modulate MDSC functional activity. Translational relevance was indicated by the finding that the same growth factors, cytokines, chemokines, and adhesion molecules responsible for the mobilization and recruitment of PMN-MDSCs into inflammatory CT26 murine tumors were also coordinately upregulated with increasing IL1ß expression in human renal cell carcinoma tumors. These studies demonstrated that IL1ß stimulated the components of a multifaceted inflammatory program that produces, mobilizes, chemoattracts, activates, and mediates the infiltration of PMN-MDSCs into inflammatory tumors to promote tumor progression.

Unfolded Protein Response Differentially Regulates TLR4-Induced Cytokine Expression in Distinct Macrophage Populations.

Cellular stress responses are often engaged at sites of inflammation and can alter macrophage cytokine production. We now report that macrophages in distinct states of differentiation or in different temporal stages of inflammatory response exhibit differential sensitivity to cell stress mediated alterations in M1-like polarized inflammatory cytokine production. Tunicamycin (Tm) treatment of bone marrow derived macrophages (BMDM) cultured with M-CSF cultured bone marrow derived macrophages (M-BMDM) had markedly amplified M1-like responses to LPS, exhibiting higher levels of IL12p40 and IL12p35 mRNAs while BMDM cultured with GM-CSF, which normally express high IL12 subunit production in response to LPS, were relatively unaltered. Anti-inflammatory IL10 mRNA production in LPS-stimulated M-BMDM was greatly reduced by cell stress. These changes in cytokine mRNA levels resulted from altered rates of transcription and mRNA decay. Stress also altered cytokine protein production. Resident liver macrophages isolated from mice treated with Tm showed elevated levels of IL12 subunit mRNA production following LPS stimulation. Furthermore, macrophages infiltrating the liver during the early phase of acetaminophen injury (24 h) had little stress-mediated change in cytokine mRNA production while cells isolated in the later phase (48-72 h) exhibited higher sensitivity for stress elevated cytokine production. Hence cultured macrophages developed using different growth/differentiation factors and macrophages from different temporal stages of injury in vivo show markedly different sensitivity to cell stress for altered inflammatory cytokine production. These findings suggest that cellular stress can be an important modulator of the magnitude and character of myeloid inflammatory activity.

Immunological Correlates of Response to Immune Checkpoint Inhibitors in Metastatic Urothelial Carcinoma.

BACKGROUND: The identification of prognostic and/or predictive biomarkers for response to immune checkpoint inhibitors (ICI) could help guide treatment decisions. OBJECTIVE: We assessed changes in programmed cell death-1 (PD1)/PD1 ligand (PDL1) expression in key immunomodulatory cell subsets (myeloid-derived suppressor cells [MDSC]; cytotoxic T lymphocytes [CTL]) following ICI therapy and investigated whether these changes correlated with outcomes in patients with metastatic urothelial carcinoma (mUC). PATIENTS AND METHODS: Serial peripheral blood samples were collected from ICI-treated mUC patients. Flow cytometry was used to quantify PD1/PDL1 expression on MDSC (CD33+HLADR-) and CTL (CD8+CD4-) from peripheral blood mononuclear cells. MDSC were grouped into monocytic (M)-MDSC (CD14+CD15-), polymorphonuclear (PMN)-MDSC (CD14-CD15+), and immature (I)-MDSC (CD14-CD15-). Mixed-model regression and Wilcoxon signed-rank or rank-sum tests were performed to assess post-ICI changes in immune biomarker expression and identify correlations between PD1/PDL1 expression and objective response to ICI. RESULTS: Of 41 ICI-treated patients, 26 received anti-PDL1 (23 atezolizumab/3 avelumab) and 15 received anti-PD1 (pembrolizumab) therapy. Based on available data, 27.5% had prior intravesical Bacillus Calmette-Guérin therapy, 42% had prior neoadjuvant chemotherapy, and 70% had prior cystectomy or nephroureterectomy. Successive doses of anti-PDL1 correlated with decreased percentage of PDL1+ (%PDL1+) M-MDSC, while doses of anti-PD1 correlated with decreased %PD1+ M- and I-MDSC. Although pre-treatment %PD1+ CTL did not predict response, a greater %PD1+ CTL within 9 weeks after ICI initiation correlated with objective response. CONCLUSIONS: Treatment with ICI correlated with distinct changes in PD1/PDL1-expressing peripheral immune cell subsets, which may predict objective response to ICI. Further studies are required to validate immune molecular expression as a prognostic and/or predictive biomarker for long-term outcomes in mUC.

Evaluation of T cell infiltration in matched biopsy and nephrectomy samples in renal cell carcinoma.

T cell infiltration in tumors has been investigated as a biomarker of response to checkpoint inhibitors. Neo-adjuvant studies in renal cell carcinoma (RCC) may provide a unique opportunity to compare T cell infiltration in a pretreatment renal mass biopsy to a posttreatment nephrectomy specimen, and thus evaluate the effects of immune checkpoint inhibitors. However, there are no data regarding the association of T cell infiltration in matched biopsy and nephrectomy samples without intervening treatment. Understanding this association will inform investigation of this potential biomarker in future studies.Matched biopsy and nephrectomy samples (without intervening systemic therapy) were identified from patients with nonmetastatic RCC. Selected tissue sections from biopsy and nephrectomy samples were reviewed and marked for intratumoral lymphocytes by a pathologist. Immunohistochemistry (IHC) was utilized to stain for T cell markers (CD3, CD4, and CD8). Intratumoral staining was then quantified in the tissue sections as counts per total tumor area surveyed. Spearman correlation (r) was used to measure associations.Thirty matched pairs were investigated. The median interval between biopsy and nephrectomy was 2.8 (0.2-87.7) months. Clear cell was the most common histology (29/30; 97%). There was a statistically significant positive correlation between the frequency of CD3 and CD8 T cells between matched biopsy and nephrectomy samples (r = 0.39; P = .036 and r = 0.38; P = .041, respectively).The frequencies of CD8+ T cells in matched biopsy and nephrectomy samples in RCC in the absence of intervening treatment have been characterized and show a positive correlation between matched biopsy and nephrectomy samples.

Active surveillance in metastatic renal-cell carcinoma: a prospective, phase 2 trial.

BACKGROUND: A subset of patients with metastatic renal-cell carcinoma show indolent growth of metastases. Because of the toxicity and non-curative nature of systemic therapy, some of these patients could benefit from initial active surveillance. We aimed to characterise the time to initiation of systemic therapy in patients with metastatic renal-cell carcinoma under active surveillance. METHODS: In this prospective phase 2 trial, we enrolled patients with treatment-naive, asymptomatic, metastatic renal-cell carcinoma from five hospitals in the USA, Spain, and the UK. Patients were radiographically assessed at baseline, every 3 months for year 1, every 4 months for year 2, then every 6 months thereafter. Patients continued on observation until initiation of systemic therapy for metastatic renal-cell carcinoma; a decision that was made at the discretion of the treating physician and patient. The primary endpoint of the study was time to initiation of systemic therapy in the per-protocol population. The follow-up of patients is ongoing. FINDINGS: Between Aug 21, 2008, and June 7, 2013, we enrolled 52 patients. Median follow-up of patients in the study was 38·1 months (IQR 29·4-48·9). In the 48 patients included in analysis, median time on surveillance from registration on study until initiation of systemic therapy was 14·9 months (95% CI 10·6-25·0). Multivariate analysis showed that higher numbers of International Metastatic Database Consortium (IMDC) adverse risk factors (p=0·0403) and higher numbers of metastatic disease sites (p=0·0414) were associated with a shorter surveillance period. 22 (46%) patients died during the study period, all from metastatic renal-cell carcinoma. INTERPRETATION: A subset of patients with metastatic renal-cell carcinoma can safely undergo surveillance before starting systemic therapy. Additional investigation is required to further define the benefits and risks of this approach. FUNDING: None.

Myeloid-derived suppressor cells adhere to physiologic STAT3- vs STAT5-dependent hematopoietic programming, establishing diverse tumor-mediated mechanisms of immunologic escape.

The receptor tyrosine kinase inhibitor, sunitinib, is astonishingly effective in its capacity to reduce MDSCs in peripheral tissues such as blood (human) and spleen (mouse), restoring responsiveness of bystander T lymphocytes to TcR stimulation. Sunitinib blocks proliferation of undifferentiated MDSCs and decreases survival of more differentiated neutrophilic MDSC (n-MDSC) progeny. Ironically, sunitinib's profound effects are observed even in a total absence of detectable anti-tumor therapeutic response. This is best explained by the presence of disparate MDSC-conditioning stimuli within individual body compartments, allowing sensitivity and resistance to sunitinib to coexist within the same mouse or patient. The presence or absence of GM-CSF is likely the major determinant in each compartment, given that GM-CSF's capacity to preempt STAT3-dependent with dominant STAT5-dependent hematopoietic programming confers sunitinib resistance and redirects differentiation from the n-MDSC lineage to the more versatile monocytoid (m-MDSC) lineage. The clinical sunitinib experience underscores that strategies for MDSC and Treg depletions must be mindful of disparities among body compartments to avoid sanctuary effects. Ironically, m-MDSCs manifesting resistance to sunitinib also have the greatest potential to differentiate into tumoricidal accessory cells, by virtue of their capacity to respond to T cell-secreted IFN-γ or to TLR agonists with nitric oxide and peroxynitrate production.

Sunitinib mediates reversal of myeloid-derived suppressor cell accumulation in renal cell carcinoma patients.

PURPOSE: Immune dysfunction reported in renal cell carcinoma (RCC) patients may contribute to tumor progression. Myeloid-derived suppressor cells (MDSC) represent one mechanism by which tumors induce T-cell suppression. Several factors pivotal to the accumulation of MDSC are targeted by the tyrosine kinase inhibitor, sunitinib. The effect of sunitinib on MDSC-mediated immunosuppression in RCC patients has been investigated. EXPERIMENTAL DESIGN: Patient peripheral blood levels of MDSC and regulatory T-cell (Treg) and T-cell production of IFN-gamma were evaluated before and after sunitinib treatment. Correlations between MDSC and Treg normalization as well as T-cell production of IFN-gamma were examined. The in vitro effect of sunitinib on patient MDSC was evaluated. RESULTS: Metastatic RCC patients had elevated levels of CD33(+)HLA-DR(-) and CD15(+)CD14(-) MDSC, and these were partially overlapping populations. Treatment with sunitinib resulted in significant reduction in MDSC measured by several criteria. Sunitinib-mediated reduction in MDSC was correlated with reversal of type 1 T-cell suppression, an effect that could be reproduced by the depletion of MDSC in vitro. MDSC reduction in response to sunitinib correlated with a reversal of CD3(+)CD4(+)CD25(hi)Foxp3(+) Treg cell elevation. No correlation existed between a change in tumor burden and a change in MDSC, Treg, or T-cell production of IFN-gamma. In vitro addition of sunitinib reduced MDSC viability and suppressive effect when used at >/=1.0 microg/mL. Sunitinib did not induce MDSC maturation in vitro. CONCLUSIONS: Sunitinib-based therapy has the potential to modulate antitumor immunity by reversing MDSC-mediated tumor-induced immunosuppression.

GD3, an overexpressed tumor-derived ganglioside, mediates the apoptosis of activated but not resting T cells.

We previously elucidated an important role for gangliosides in renal cell carcinoma-mediated T lymphocyte apoptosis, although the mechanism by which they mediated lymphocyte death remained unclear. Here, we show that when added in purified form, GD3 is internalized by activated T cells, initiating a series of proapoptotic events, including the induction of reactive oxygen species (ROS), an enhancement of p53 and Bax accumulation, an increase in mitochondrial permeability, cytochrome c release, and the activation of caspase-9. GD3-induced apoptosis of activated T cells was dose dependent and inhibitable by pretreating the lymphocytes with N-acetylcysteine, cyclosporin A, or bongkrekic acid, emphasizing the essential role of ROS and mitochondrial permeability to the process. Ganglioside-induced T-cell killing was associated with the caspase-dependent degradation of nuclear factor-kappaB-inducible, antiapoptotic proteins, including RelA; this suggests that their loss is initiated only after the cascade is activated and that their disappearance amplifies but not triggers GD3 susceptibility. Resting T cells did not internalize appreciable levels of GD3 and did not undergo any of the proapoptotic changes that characterize activated T lymphocytes exposed to the ganglioside. RelA overexpression endows Jurkat cells with resistance to GD3-mediated apoptosis, verifying the role of the intact transcription factor in mediating protection from the ganglioside.