Improvement of tumor neoantigen detection by high field asymmetric waveform ion mobility mass spectrometry.

Cancer neoantigens have been shown to elicit cancer-specific T-cell responses and have garnered much attention for their roles in both spontaneous and therapeutically induced antitumor responses. Mass spectrometry (MS) profiling of tumor immunopeptidomes has been used, in part, to identify MHC-bound mutant neoantigen ligands. However, under standard conditions, MS-based detection of such rare but clinically relevant neoantigens is relatively insensitive, requiring 300 million cells or more. Here, to quantitatively define the minimum detectable amounts of therapeutically relevant MHC-I and MHC-II neoantigen peptides, we analyzed different dilutions of immunopeptidomes isolated from the well-characterized T3 mouse methylcholanthrene (MCA)-induced cell line by MS. Using either data-dependent acquisition (DDA) or parallel reaction monitoring (PRM), we established the minimum amount of material required to detect the major T3 neoantigens in the presence or absence of high field asymmetric waveform ion mobility spectrometry (FAIMS). This analysis yielded a 14-fold enhancement of sensitivity in detecting the major T3 MHC-I neoantigen (mLama4) with FAIMS-PRM compared with PRM without FAIMS, allowing ex-vivo detection of this neoantigen from an individual 100 mg T3 tumor. These findings were then extended to two other independent MCA-sarcoma lines (1956 and F244). This study demonstrates that FAIMS substantially increases the sensitivity of MS-based characterization of validated neoantigens from tumors.

VISTA checkpoint inhibition by pH-selective antibody SNS-101 with optimized safety and pharmacokinetic profiles enhances PD-1 response.

VISTA, an inhibitory myeloid-T-cell checkpoint, holds promise as a target for cancer immunotherapy. However, its effective targeting has been impeded by issues such as rapid clearance and cytokine release syndrome observed with previous VISTA antibodies. Here we demonstrate that SNS-101, a newly developed pH-selective VISTA antibody, addresses these challenges. Structural and biochemical analyses confirmed the pH-selectivity and unique epitope targeted by SNS-101. These properties confer favorable pharmacokinetic and safety profiles on SNS-101. In syngeneic tumor models utilizing human VISTA knock-in mice, SNS-101 shows in vivo efficacy when combined with a PD-1 inhibitor, modulates cytokine and chemokine signaling, and alters the tumor microenvironment. In summary, SNS-101, currently in Phase I clinical trials, emerges as a promising therapeutic biologic for a wide range of patients whose cancer is refractory to current immunotherapy regimens.

Highly immunogenic cancer cells require activation of the WNT pathway for immunological escape.

PD-1 blockade exerts antitumor effects by reinvigorating tumor antigen­specific CD8+ T cells. Whereas neoantigens arising from gene alterations in cancer cells comprise critical tumor antigens in antitumor immunity, a subset of non­small cell lung cancers (NSCLCs) harboring substantial tumor mutation burden (TMB) lack CD8+ T cells in the tumor microenvironment (TME), which results in resistance to PD-1 blockade therapy. To overcome this resistance, clarifying the mechanism(s) impairing antitumor immunity in highly mutated NSCLCs is an urgent issue. Here, we showed that activation of the WNT/ß-catenin signaling pathway contributed to the development of a noninflamed TME in tumors with high TMB. NSCLCs that lacked immune cell infiltration into the TME despite high TMB preferentially up-regulated the WNT/ß-catenin pathway. Immunologic assays revealed that those patients harbored neoantigen-specific CD8+ T cells in the peripheral blood but not in the TME, suggesting impaired T cell infiltration into the TME due to the activation of WNT/ß-catenin signaling. In our animal models, the accumulation of gene mutations in cancer cells increased CD8+ T cell infiltration into the TME, thus slowing tumor growth. However, further accumulation of gene mutations blunted antitumor immunity by excluding CD8+ T cells from tumors in a WNT/ß-catenin signaling-dependent manner. Combined treatment with PD-1 blockade and WNT/ß-catenin signaling inhibitors induced better antitumor immunity than either treatment alone. Thus, we propose a mechanism-oriented combination therapy whereby immune checkpoint inhibitors can be combined with drugs that target cell-intrinsic oncogenic signaling pathways involved in tumor immune escape.

Unexpected suppression of tumorigenesis by c-MYC via TFAP4-dependent restriction of stemness in B lymphocytes.

The proliferative burst of B lymphocytes is essential for antigen receptor repertoire diversification during the development and selective expansion of antigen-specific clones during immune responses. High proliferative activity inevitably promotes oncogenesis, the risk of which is further elevated in B lymphocytes by endogenous gene rearrangement and somatic mutations. However, B-cell-derived cancers are rare, perhaps owing to putative intrinsic tumor-suppressive mechanisms. We show that c-MYC facilitates B-cell proliferation as a protumorigenic driver and unexpectedly coengages counteracting tumor suppression through its downstream factor TFAP4. TFAP4 is mutated in human lymphoid malignancies, particularly in >10% of Burkitt lymphomas, and reduced TFAP4 expression was associated with poor survival of patients with MYC-high B-cell acute lymphoblastic leukemia. In mice, insufficient TFAP4 expression accelerated c-MYC-driven transformation of B cells. Mechanistically, c-MYC suppresses the stemness of developing B cells by inducing TFAP4 and restricting self-renewal of proliferating B cells. Thus, the pursuant transcription factor cascade functions as a tumor suppressor module that safeguards against the transformation of developing B cells.

The PD-1 expression balance between effector and regulatory T cells predicts the clinical efficacy of PD-1 blockade therapies.

Immune checkpoint blockade has provided a paradigm shift in cancer therapy, but the success of this approach is very variable; therefore, biomarkers predictive of clinical efficacy are urgently required. Here, we show that the frequency of PD-1+CD8+ T cells relative to that of PD-1+ regulatory T (Treg) cells in the tumor microenvironment can predict the clinical efficacy of programmed cell death protein 1 (PD-1) blockade therapies and is superior to other predictors, including PD ligand 1 (PD-L1) expression or tumor mutational burden. PD-1 expression by CD8+ T cells and Treg cells negatively impacts effector and immunosuppressive functions, respectively. PD-1 blockade induces both recovery of dysfunctional PD-1+CD8+ T cells and enhanced PD-1+ Treg cell-mediated immunosuppression. A profound reactivation of effector PD-1+CD8+ T cells rather than PD-1+ Treg cells by PD-1 blockade is necessary for tumor regression. These findings provide a promising predictive biomarker for PD-1 blockade therapies.

TREM2 Modulation Remodels the Tumor Myeloid Landscape Enhancing Anti-PD-1 Immunotherapy.

Checkpoint immunotherapy unleashes T cell control of tumors, but is undermined by immunosuppressive myeloid cells. TREM2 is a myeloid receptor that transmits intracellular signals that sustain microglial responses during Alzheimer's disease. TREM2 is also expressed by tumor-infiltrating macrophages. Here, we found that Trem2-/- mice are more resistant to growth of various cancers than wild-type mice and are more responsive to anti-PD-1 immunotherapy. Furthermore, treatment with anti-TREM2 mAb curbed tumor growth and fostered regression when combined with anti-PD-1. scRNA-seq revealed that both TREM2 deletion and anti-TREM2 are associated with scant MRC1+ and CX3CR1+ macrophages in the tumor infiltrate, paralleled by expansion of myeloid subsets expressing immunostimulatory molecules that promote improved T cell responses. TREM2 was expressed in tumor macrophages in over 200 human cancer cases and inversely correlated with prolonged survival for two types of cancer. Thus, TREM2 might be targeted to modify tumor myeloid infiltrates and augment checkpoint immunotherapy.

Blockade of EGFR improves responsiveness to PD-1 blockade in EGFR-mutated non-small cell lung cancer.

The clinical efficacy of anti-PD-1 (programmed cell death-1) monoclonal antibody (mAb) against cancers with oncogenic driver gene mutations, which often harbor a low tumor mutation burden, is variable, suggesting different contributions of each driver mutation to immune responses. Here, we investigated the immunological phenotypes in the tumor microenvironment (TME) of epidermal growth factor receptor (EGFR)-mutated lung adenocarcinomas, for which anti-PD-1 mAb is largely ineffective. Whereas EGFR-mutated lung adenocarcinomas had a noninflamed TME, CD4+ effector regulatory T cells, which are generally present in the inflamed TME, showed high infiltration. The EGFR signal activated cJun/cJun N-terminal kinase and reduced interferon regulatory factor-1; the former increased CCL22, which recruits CD4+ regulatory T cells, and the latter decreased CXCL10 and CCL5, which induce CD8+ T cell infiltration. The EGFR inhibitor erlotinib decreased CD4+ effector regulatory T cells infiltration in the TME and in combination with anti-PD-1 mAb showed better antitumor effects than either treatment alone. Our results suggest that EGFR inhibitors when used in conjunction with anti-PD-1 mAb could increase the efficacy of immunotherapy in lung adenocarcinomas.

Tyrosine kinase inhibitor imatinib augments tumor immunity by depleting effector regulatory T cells.

This report addresses whether small molecules can deplete FoxP3-expressing regulatory T (T reg) cells, thereby augmenting antitumor immunity. Imatinib, a tyrosine kinase inhibitor of oncogenic BCR-ABL protein expressed by chronic myelogenous leukemia (CML) cells, possesses off-targets including LCK expressed in T cells. We showed that imatinib-treated CML patients in complete molecular remission (CMR) exhibited selective depletion of effector T reg (eT reg) cells and significant increase in effector/memory CD8+ T cells while non-CMR patients did not. Imatinib at CML-therapeutic concentrations indeed induced apoptosis specifically in eT reg cells and expanded tumor antigen-specific CD8+ T cells in vitro in healthy individuals and melanoma patients, and suppressed colon tumor growth in vivo in mice. Mechanistically, because of FoxP3-dependent much lower expression of LCK and ZAP-70 in T reg cells compared with other T cells, imatinib inhibition of LCK further reduced their TCR signal intensity, rendering them selectively susceptible to signal-deprived apoptotis. Taken together, eT reg cell depletion by imatinib is instrumental in evoking effective immune responses to various cancers.

Clinical response to PD-1 blockade correlates with a sub-fraction of peripheral central memory CD4+ T cells in patients with malignant melanoma.

Cancer immunotherapy that blocks immune checkpoint molecules, such as PD-1/PD-L1, unleashes dysfunctional antitumor T-cell responses and has durable clinical benefits in various types of cancers. Yet its clinical efficacy is limited to a small proportion of patients, highlighting the need for identifying biomarkers that can predict the clinical response by exploring antitumor responses crucial for tumor regression. Here, we explored comprehensive immune-cell responses associated with clinical benefits using PBMCs from patients with malignant melanoma treated with anti-PD-1 monoclonal antibody. Pre- and post-treatment samples were collected from two different cohorts (discovery set and validation set) and subjected to mass cytometry assays that measured the expression levels of 35 proteins. Screening by high dimensional clustering in the discovery set identified increases in three micro-clusters of CD4+ T cells, a subset of central memory CD4+ T cells harboring the CD27+FAS-CD45RA-CCR7+ phenotype, after treatment in long-term survivors, but not in non-responders. The same increase was also observed in clinical responders in the validation set. We propose that increases in this subset of central memory CD4+ T cells in peripheral blood can be potentially used as a predictor of clinical response to PD-1 blockade therapy in patients with malignant melanoma.

Roles of regulatory T cells in cancer immunity.

CD4(+) regulatory T cells (Tregs) expressing the transcription factor FoxP3 are highly immune suppressive and play central roles in the maintenance of self-tolerance and immune homeostasis, yet in malignant tumors they promote tumor progression by suppressing effective antitumor immunity. Indeed, higher infiltration by Tregs is observed in tumor tissues, and their depletion augments antitumor immune responses in animal models. Additionally, increased numbers of Tregs and, in particular, decreased ratios of CD8(+) T cells to Tregs among tumor-infiltrating lymphocytes are correlated with poor prognosis in various types of human cancers. The recent success of cancer immunotherapy represented by immune checkpoint blockade has provided a new insight in cancer treatment, yet more than half of the treated patients did not experience clinical benefits. Identifying biomarkers that predict clinical responses and developing novel immunotherapies are therefore urgently required. Cancer patients whose tumors contain a large number of neoantigens stemming from gene mutations, which have not been previously recognized by the immune system, provoke strong antitumor T-cell responses associated with clinical responses following immune checkpoint blockade, depending on the resistance to Treg-mediated suppression. Thus, integration of a strategy restricting Treg-mediated immune suppression may expand the therapeutic spectrum of cancer immunotherapy towards patients with a lower number of neoantigens. In this review, we address the current understanding of Treg-mediated immune suppressive mechanisms in cancer, the involvement of Tregs in cancer immunotherapy, and strategies for effective and tolerable Treg-targeted therapy.

Favorable response to trastuzumab plus irinotecan combination therapy in two patients with HER2-positive relapsed small-cell lung cancer.

Small-cell lung cancer (SCLC) easily recurs with multidrug resistance phenotype. However, standard therapeutic strategies for relapsed-SCLC remain unestablished. Human epidermal growth factor receptor 2 (HER2) expression correlates with poor prognosis in extensive disease-SCLC. We have reported previously that HER2 expression is upregulated when HER2-positive SCLC cells acquire chemoresistance, and also demonstrated that trastuzumab exerts significant antitumor activity toward HER2-upregulated chemoresistant SCLC, mainly via antibody-dependent cell-mediated cytotoxicity mechanism. Based on these preclinical data, we treated two patients with HER2-positive SCLC by combination of trastuzumab (6 mg/kg, day 1) and irinotecan (80 mg/m(2), days 1 and 8) every 21 days as the third-line chemotherapy following two prior regimens, first-line carboplatin plus etoposide and second-line amrubicin. One patient achieved partial response after the first cycle and received 6 cycles in total without disease progression for 4.5 months. The other also received 4 cycles and kept stable disease for 3.5 months. This treatment can be continued safely at an outpatient clinic without any severe adverse event. In conclusion, trastuzumab plus irinotecan chemotherapy is promising and feasible against HER2-positive relapsed SCLC. Further clinical studies are encouraged to confirm the antitumor efficacy of trastuzumab in SCLC.

Phase II trial of pemetrexed continuation--maintenance after carboplatin-based induction in untreated non-squamous non-small cell lung cancer.

AIM: We conducted a phase II study to evaluate the efficacy and safety of pemetrexed continuation-maintenance after carboplatin-based induction for advanced non-squamous non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Thirty-four patients with advanced or recurrent non-squamous NSCLC received carboplatin (area under the concentration-time curve 6 mg/ml×min) plus pemetrexed (500 mg/m(2)) on day 1 tri-weekly. After four cycles of induction, patients without disease progression received pemetrexed maintenance until disease progression or unacceptable toxicity. RESULTS: Twenty-five patients completed induction and 22 received maintenance. The 1-year survival, objective response and disease control rates were 70.3%, 32.4% and 88.2%, respectively. The median progression-free survival and overall survival of all patients were 5.2 and 23.3 months. The incidental rates of grade 3 or more severe adverse events were low. CONCLUSION: This regimen appears to be an appropriate option for chemonaïve patients with advanced non-squamous NSCLC.

Phase II study of pemetrexed plus intermittent erlotinib combination therapy for pretreated advanced non-squamous non-small cell lung cancer with documentation of epidermal growth factor receptor mutation status.

INTRODUCTION: Erlotinib and pemetrexed have been approved for the second-line and maintenance treatment of non-small cell lung cancer (NSCLC). With the recommended doses determined by our previous phase I study, we conducted a phase II study to evaluate the efficacy and safety of combination of the two agents in pretreated non-squamous NSCLC patients. METHODS: This study was performed in patients with stage IIIB/IV or post-surgically recurrent non-squamous NSCLC whose disease had progressed on or after receiving first-line chemotherapy. Patients received 500 mg/m(2) of intravenous pemetrexed every 21 days and 150 mg of oral erlotinib on days 2-16 until disease progression, unacceptable toxicity, or withdrawal of consent. The expected response rate and threshold were defined as 33.5% and 10%, respectively. Assuming a one-sided alpha of 5%, a power of 80%, the possible deviation from assessment, 26 patients were necessary. RESULTS: A total of 27 patients, 16 males and 11 females were recruited. Patients had the median age of 70 years (range, 48-80 years) and included 21 stage IV diseases, 22 adenocarcinomas. Epidermal growth factor receptor (EGFR) mutations were examined in all patients. One patient had positive EGFR mutation, but the other 26 patients had wild-type EGFR. The median number of treatment courses was 3 (range, 1 to over 19). The best overall response rate and disease control rate were 11.1% and 63.0%, respectively. The median progression-free survival and overall survival were 2.8 months (95% confidence interval (CI); 1.9-7.5 months) and 15.8 months (95% CI; 9.3 months to not available), respectively. Dermal, hepatic, gastrointestinal and hematological disorders were the frequently observed adverse events. One patient experienced grade 3 drug-induced interstitial lung disease. CONCLUSIONS: We could not demonstrate the add-on effect of intermittent erlotinib on pemetrexed in a second-line setting for patients with non-squamous NSCLC without EGFR mutations.

Emergency department visits after hours by lung cancer patients in Japan.

PURPOSE: The aims of this study were to clarify frequency with which Japanese lung cancer patients visited an emergency department (ED) after hours and their final outcome. METHODS: This is a retrospective and single institutional study. We reviewed medical records of patients who died of lung cancer from January 2008 to June 2012 at Osaka Police Hospital who had been followed up since diagnosis of lung cancer until death. We compared patients who had visited the ED after hours on weekdays, weekends, or holidays over their lives with cancer (ED visitors) and patients who had never visited the ED (non-ED visitors). RESULTS: Overall, 245 patients met the inclusion criteria for analysis. There were 149 after hours ED visits by 106 lung cancer patients. Mean number of ED visits was 0.6 for all patients. Median interval from ED visit to death was 49 days. The most common chief compliant for these patients was respiratory problems (37.6%). Most patients visited the ED during chemotherapy (32.9%) or for best supportive care (42.3%). Directly after ED visits, 56.4% of ED visitors were finally hospitalized. In a multivariate analysis, performance status (PS) (odds ratio [OR]: 11.2, 95% confidence interval [CI]: 2.1-59.0, p = 0.004) and cancer stage (OR: 0.003, 95% CI: 0.0006-0.014, p < 0.001) at diagnosis were statistically associated with ED visits after hours. CONCLUSIONS: Japanese patients with lung cancer frequently visit ED after hours. An ED visit is itself an indicator of poor prognosis.

Combination chemotherapy with intermittent erlotinib and pemetrexed for pretreated patients with advanced non-small cell lung cancer: a phase I dose-finding study.

BACKGROUND: Erlotinib and pemetrexed have been approved for the second-line treatment of non-small cell lung cancer (NSCLC). These two agents have different mechanisms of action. Combined treatment with erlotinib and pemetrexed could potentially augment the antitumor activity of either agent alone. In the present study, we investigated the safety profile of combined administration of the two agents in pretreated NSCLC patients. METHODS: A phase I dose-finding study (Trial registration: UMIN000002900) was performed in patients with stage III/IV nonsquamous NSCLC whose disease had progressed on or after receiving first-line chemotherapy. Patients received 500 mg/m(2) of pemetrexed intravenously every 21 days and erlotinib (100 mg at Level 1 and 150 mg at Level 2) orally on days 2-16. RESULTS: Twelve patients, nine males and three females, were recruited. Patient characteristics included a median age of 66 years (range, 48-78 years), stage IV disease (nine cases), adenocarcinoma (seven cases) and activating mutation-positives in the epidermal growth factor receptor gene (two cases). Treatment was well-tolerated, and the recommended dose of erlotinib was fixed at 150 mg. Dose-limiting toxicities were experienced in three patients and included: grade 3 elevation of serum alanine aminotransferase, repetitive grade 4 neutropenia that required reduction of the second dose of pemetrexed and grade 3 diarrhea. No patient experienced drug-induced interstitial lung disease. Three patients achieved a partial response and stable disease was maintained in five patients. CONCLUSIONS: Combination chemotherapy of intermittent erlotinib with pemetrexed was well-tolerated, with promising efficacy against pretreated advanced nonsquamous NSCLC.