Clinical relevance of PD-1 positive CD8 T-cells in gastric cancer.

BACKGROUND: We evaluated the relevance of PD-1+CD8+ T-cells in gastric cancer (GC) including prognostic significance, association with chemotherapy and immunotherapy sensitivity and correlations with the tumor microenvironment (TME). METHODS: Discovery cohort: GC samples were evaluated for AE1/3, CD8, PD-1, Ki-67 and Granzyme-B expression with fluorescence-based multiplex immunohistochemistry (mIHC). Validation cohorts: we analyzed bulk RNAseq GC datasets from TCGA, the "3G" chemotherapy trial and an immunotherapy phase 2 trial. The cox proportional hazards model was used to identify factors that influenced overall survival (OS). To study the TME, we analyzed single-cell RNAseq performed on GCs. RESULTS: In the discovery cohort of 350 GCs, increased PD-1 expression of CD8 T-cells was prognostic for OS (HR 0.822, p = 0.042). PD-1 expression in CD8 T-cells highly correlated with cytolytic [Granzyme-B+] (r = 0.714, p < 0.001) and proliferative [Ki-67+] (r = 0.798, p < 0.001) activity. Analysis of bulk RNAseq datasets showed tumors with high PD-1 and CD8A expression levels had improved OS when treated with immunotherapy (HR 0.117, p = 0.036) and chemotherapy (HR 0.475, p = 0.017). Analysis of an scRNAseq dataset of 152,423 cells from 40 GCs revealed that T-cell and NK-cell proportions were higher (24% vs 18% and 19% vs 15%, p < 0.0001), while macrophage proportions were lower (7% vs 11%, p < 0.0001) in CD8PD-1high compared to CD8PD-1low tumors. CONCLUSION: This is one of the largest GC cohorts of mIHC combined with analysis of multiple datasets providing orthogonal validation of the clinical relevance of PD-1+CD8+ T-cells being associated with improved OS. CD8PD-1high tumors have distinct features of an immunologically active, T-cell inflamed TME.

Immunohistochemistry study of tumor vascular normalization and anti-angiogenic effects of sunitinib versus bevacizumab prior to dose-dense doxorubicin/cyclophosphamide chemotherapy in HER2-negative breast cancer.

PURPOSE: Tumor angiogenesis controlled predominantly by vascular endothelial growth factor and its receptor (VEGF-VEGFR) interaction plays a key role in the growth and propagation of cancer cells. However, the newly formed network of blood vessels is disorganized and leaky. Pre-treatment with anti-angiogenic agents can "normalize" the tumor vasculature allowing effective intra-tumoral delivery of standard chemotherapy. Immunohistochemistry (IHC) analysis was applied to investigate and compare the vascular normalization and anti-angiogenic effects of two commonly used anti-angiogenic agents, Sunitinib and Bevacizumab, administered prior to chemotherapy in HER2-negative breast cancer patients. METHODS: This prospective clinical trial enrolled 38 patients into a sunitinib cohort and 24 into a bevacizumab cohort. All received 4 cycles of doxorubicin/cyclophosphamide chemotherapy and pre-treatment with either sunitinib or bevacizumab. Tumor biopsies were obtained at baseline, after cycle 1 (C1) and cycle 4 (C4) of chemotherapy. IHC was performed to assess the tumor vascular normalization index (VNI), lymphatic vessel density (LVD), Ki67 proliferation index and expression of tumor VEGFR2. RESULTS: In comparison to Bevacizumab, Sunitinib led to a significant increase in VNI post-C1 and C4 (p < 0.001 and 0.001) along with decrease in LVD post-C1 (p = 0.017). Both drugs when combined with chemotherapy resulted in significant decline in tumor proliferation after C1 and C4 (baseline vs post-C4 Ki67 index p = 0.006 for Sunitinib vs p = 0.021 for Bevacizumab). Bevacizumab resulted in a significant decrease in VEGFR2 expression post-C1 (p = 0.004). CONCLUSION: Sunitinib, in comparison to Bevacizumab showed a greater effect on tumor vessel modulation and lymphangiogenesis suggesting that its administration prior to chemotherapy might result in improved drug delivery. TRIAL REGISTRY: ClinicalTrials.gov: NCT02790580 (first posted June 6, 2016).

Outcomes of a Phase II Study of Intraperitoneal Paclitaxel plus Systemic Capecitabine and Oxaliplatin (XELOX) for Gastric Cancer with Peritoneal Metastases.

BACKGROUND: Adding intraperitoneal paclitaxel (IP-PTX) to paclitaxel/5-fluoropyrimidine has shown promising results in patients with gastric cancer peritoneal metastases (GCPM) but has not been studied with standard-of-care platinum/fluoropyrimidine combinations. Our goal to was evaluate IP-PTX with capecitabine/oxaliplatin (XELOX) in GCPM. METHODS: Forty-four patients with GCPM received IP PTX (40 mg/m2, Days 1, 8), oral capecitabine (1000 mg/m2 twice daily, Days 1-14) and intravenous oxaliplatin (100 mg/m2, Day 1) in 21-day cycles. Patients with synchronous GCPM underwent conversion surgery if they had good response after chemotherapy, conversion to negative cytology, no extraperitoneal metastasis, and no peritoneal disease during surgery. The primary endpoint was overall survival and secondary endpoints were progression-free survival and safety. Outcomes from the trial were compared against a matched cohort of 39 GCPM patients who received systemic chemotherapy (SC) comprising platinum/fluoropyrimidine. RESULTS: The median OS for the IP and SC groups was 14.6 and 10.6 months (hazard ratio [HR] 0.44; 95% confidence interval [CI], 0.26-0.74; p = 0.002). The median PFS for the IP and SC group was 9.5 and 4.4 months respectively (HR 0.39; 95% CI 0.25-0.66; p < 0.001). Patients in the SC group were younger (IP vs. SC, 61 vs. 56 years, p = 0.021) and had better performance status (ECOG 0, IP vs. SC, 47.7% vs. 76.9%, p = 0.007) compared with the IP cohort. In IP group, conversion surgery was performed in 36.1% (13/36) of patients, with a median OS of 24.2 (95% CI 13.1-35.3) months and 1-year OS of 84.6%. CONCLUSIONS: IP PTX with XELOX is a promising treatment option for GCPM patients. In patients with good response, conversion surgery was feasible with favourable outcomes.

Low-Dose Nivolumab in Renal Cell Carcinoma: A Real-World Experience.

BACKGROUND: The approved doses of the single agent nivolumab - an anti-programmed cell death protein 1 (PD-1) monoclonal antibody - for renal cell carcinoma (RCC) are 3 mg/kg and a 240-mg flat dose, despite efficacy shown at lower doses in earlier CheckMate trials. In view of financial constraints, the minimum dose of nivolumab required for efficacy remains a critical area of inquiry. METHODS: A retrospective review of RCC patients receiving single-agent anti-PD-1 treatment was conducted. Using the median cutoff of the maximum dose per body weight received, we investigated the effect of lower dosages on overall survival (OS), progression-free survival (PFS), objective response rate (ORR), and immune-related adverse event-free survival (irAE-FS). Survival analysis was made by Kaplan-Meier, by uni- and multivariable Cox models, and by modeling the statistical interaction between dosages and survival. RESULTS: 32 patients were recruited: 8 patients (25%) receiving first-line treatment and 24 (75%) receiving second-line treatment and beyond. A median split at 2.15 mg/kg yielded 16 patients in both the lower-dose (LD) and the higher-dose (HD) cohort. Hazard ratios (HRs) demonstrated no difference in OS after adjustment for gender (HR = 0.22, 95% CI 0.05-1.05, p = 0.054; favoring LD), as well as in PFS after adjustment for gender and concurrent radiation therapy (HR = 0.58, 95% CI 0.25-1.34, p = 0.210; favoring LD). No differences in ORR were observed (50.0 vs. 43.8%, p = 1.00, in the LD and the HD cohort, respectively). Immune-related phenomena were observed in the LD group, including pseudoprogression and increased all-grade immune-related toxicities (irAE-FS: HR = 1.72, 95% CI 0.48-6.14, p = 0.293; favoring HD). Iterative dichotomization of dosages showed no dose-OS or dose-irAE-FS relationship. CONCLUSION: Our study suggests no apparent reduction in efficacy when using a low-dosage nivolumab regimen.

Serial Tumor Molecular Profiling of Newly Diagnosed HER2-Negative Breast Cancers During Chemotherapy in Combination with Angiogenesis Inhibitors.

BACKGROUND: Breast cancers are heterogeneous with variable clinical courses and treatment responses. OBJECTIVE: We sought to evaluate dynamic changes in the molecular landscape of HER2-negative tumors treated with chemotherapy and anti-angiogenic agents. PATIENTS AND METHODS: Newly diagnosed HER2-negative breast cancer patients received low-dose sunitinib or bevacizumab prior to four 2-weekly cycles of dose-dense doxorubicin and cyclophosphamide. Tumor biopsies were obtained at baseline, after 2 weeks and after 8 weeks of chemotherapy. Next-generation sequencing was performed to assess for single nucleotide variants (SNVs) and copy number alterations (CNAs) of 440 cancer-related genes (ACTOnco®). Observed genomic changes were correlated with the Miller-Payne histological response to treatment. RESULTS: Thirty-four patients received sunitinib and 18 received bevacizumab. In total, 77% were hormone receptor positive (HER2-/HR+) and 23% were triple negative breast cancers (TNBC). New therapy-induced mutations were infrequent, occurring only in 13%, and appeared early after a single cycle of treatment. Seventy-two percent developed changes in the variant allele frequency (VAF) of pathogenic SNVs; the majority (51%) of these changes occurred early at 2 weeks and were sustained for 8 weeks. Changes in VAF of SNVs were most commonly seen in the PI3K/mTOR/AKT pathway; 13% developed changes in pathogenic mutations, which potentially confer sensitivity to PIK3CA inhibitors. Tumors with poor Miller-Payne response to treatment were less likely to experience changes in VAF of SNVs compared with those with good response (50% [7/14] vs 15% [4/24] had no changes observed at any timepoint, p = 0.029). CONCLUSIONS: Serial molecular profiling identifies early therapy-induced genomic alterations, which may guide future selection of targeted therapies in breast cancer patients who progress after standard chemotherapy. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov: NCT02790580 (first posted June 6, 2016).

Phase Ib/II Dose Expansion Study of Lenvatinib Combined with Letrozole in Postmenopausal Women with Hormone Receptor-Positive Breast Cancer.

PURPOSE: RET is an estrogen response gene with preclinical studies demonstrating cross-talk between the RET and estrogen receptor (ER) pathways. We investigate the role of lenvatinib, a multikinase inhibitor with potent activity against RET, in patients with metastatic breast cancer. PATIENTS AND METHODS: Patients with advanced ER+/HER2- breast cancer were treated with lenvatinib plus letrozole in a phase Ib/II trial. Primary objectives included safety and recommended phase II dose (RP2D) determination in phase Ib, and objective response rates (ORR) in phase II dose expansion. RESULTS: Sixteen patients were recruited in dose finding, where deescalating doses of lenvatinib from 20 to 14 mg were investigated. Lenvatinib 14 mg plus letrozole 2.5 mg daily was determined as RP2D. Thirty-one patients with 5 median lines of prior therapy in the metastatic setting (range, 0-11) were recruited in dose expansion. In this cohort, ORR was 23.3% [95% confidence interval (CI) 9.9%-42.3%], with median duration of response (DoR) of 6.9 months [interquartile range (IQR) 5.9 to 13.1]. Clinical benefit rate ≥6 months (CBR) was 50.0% (95% CI, 31.3%-68.7%). Similar efficacy was observed in the subgroup of 25 patients who progressed on prior CDK4/6 inhibitor therapy [ORR 20.0% (95% CI, 6.8%-40.7%), median DoR 6.9 months (IQR 5.9-13.1), and CBR 52.0% (95% CI, 31.3%-72.2%)]. Pharmacodynamic studies showed target modulation, with paired tumor biopsies indicating downregulation of RET/pERK and improved vascular normalization index. CONCLUSIONS: Lenvatinib plus letrozole had manageable toxicity, with target engagement and preliminary antitumor activity observed, supporting further assessment in randomized studies.

PIPAC-OX: A Phase I Study of Oxaliplatin-Based Pressurized Intraperitoneal Aerosol Chemotherapy in Patients with Peritoneal Metastases.

PURPOSE: Pressurized intraperitoneal aerosol chemotherapy (PIPAC) is a novel laparoscopic, intraperitoneal chemotherapy delivery technique aiming to improve drug distribution and tissue penetration to treat peritoneal metastases. Thus far, PIPAC oxaliplatin is conducted at an arbitrary dose of 92 mg/m2. We conducted a phase I study to establish safety and tolerability. PATIENTS AND METHODS: We used a 3+3 dose-escalation design of PIPAC oxaliplatin for patients with peritoneal metastases from gastrointestinal tumors, after failure of at least first-line chemotherapy. Dose levels were planned at 45, 60, 90, and 120 mg/m2. RESULTS: This study included 16 patients with 24 PIPAC procedures (8 gastric; 5 colorectal; and 1 gallbladder, pancreas, and appendix cancer each). Median age and peritoneal cancer index (PCI) score were 62 years and 17, respectively. Two patients developed pancreatitis (grade 2 and 3) at 45 mg/m2, necessitating cohort expansion. Another patient developed grade 2 pancreatitis at 90 mg/m2. There were no other dose-limiting toxicities, and the highest-dose cohort (120 mg/m2) tolerated PIPAC well. Pharmacokinetic analyses demonstrated good linearity between dose and maximum concentration (r 2 = 0.95) and AUC (r 2 = 0.99). On the basis of RECIST, 62.5% and 50% had stable disease after one and two PIPAC procedures, respectively. A total of 8 patients underwent two PIPAC procedures, with improvement of median PCI and peritoneal regression grade score from 15 to 12 and 2.5 to 2.0, respectively. CONCLUSIONS: The recommended phase II dose is 120 mg/m2. Future studies should further delineate the efficacy and role of PIPAC oxaliplatin for peritoneal metastases.See related commentary by de Jong et al., p. 1830.

Safety, pharmacokinetics and tissue penetration of PIPAC paclitaxel in a swine model.

INTRODUCTION: Peritoneal carcinomatosis is difficult to treat. Pressurized Intra-Peritoneal Aerosolised Chemotherapy (PIPAC) is a novel method of delivering chemotherapy to the peritoneal cavity, aiming for homogenous and deeper drug distribution. To date, limited chemotherapeutics have been used with promising results. Here, we evaluate the pharmacokinetics, peritoneal tissue drug concentration, penetration, and short-term safety of PIPAC using solvent-based paclitaxel in swine to guide clinical trials. MATERIALS AND METHODS: PIPAC solvent-based paclitaxel was administered at 60, 30, and 15mg/m2 for 3 cohorts. Each PIPAC procedure was followed by intravenous (IV) administration of the same dose of solvent-based paclitaxel on Day 7, serving as control for pharmacokinetic comparison in the same pig. Safety and toxicity were evaluated by clinical assessment, blood counts and biochemistry. Blood samples were taken for pharmacokinetic analysis. Peritoneal biopsies were taken to measure tissue paclitaxel concentrations and distribution. RESULTS: 12 Yorkshire x Landrace pigs underwent trial procedures. With PIPAC, there was linear pharmacokinetics and lower systemic exposure to paclitaxel compared to IV administration. MALDI-MSI demonstrated concentration of paclitaxel at the peritoneal surface, with estimated 2 mm penetration. PIPAC paclitaxel had favorable toxicity profile. The most significant adverse event was neutropenia which was dose dependent, with absolute neutrophil count <1.0 × 103/µL seen at the highest dose. One pig developed grade 2 hypersensitivity reaction during IV infusion and one death occurred during the PIPAC procedure, likely from anaphylaxis; these are known potential adverse events mandating standard precautions and monitoring. CONCLUSION: PIPAC paclitaxel at 15mg/m2 may be considered for a Phase I study.

Phase I Trial of Expanded, Activated Autologous NK-cell Infusions with Trastuzumab in Patients with HER2-positive Cancers.

PURPOSE: Natural killer (NK) cells exert antibody-dependent cell cytotoxicity (ADCC). We infused expanded, activated autologous NK cells to potentiate trastuzumab-mediated ADCC in patients with HER2-positive malignancies. PATIENTS AND METHODS: In a phase I trial, patients with treatment-refractory HER2-positive solid tumors received trastuzumab, with or without bevacizumab, and autologous NK cells expanded by 10-day coculture with K562-mb15-41BBL cells. Primary objectives included safety and recommended phase II dose determination; secondary objectives included monitoring NK-cell activity and RECIST antitumor efficacy. RESULTS: In 60 cultures with cells from 31 subjects, median NK-cell expansion from peripheral blood was 340-fold (range, 91-603). NK cells expressed high levels of CD16, the mediator of ADCC, and exerted powerful killing of trastuzumab-targeted cells. In the 22 subjects enrolled in phase I dose escalation, trastuzumab plus NK cells were well tolerated; MTD was not reached. Phase IB (n = 9) included multiple cycles of NK cells (1 × 107/kg) and addition of bevacizumab. Although no objective response was observed, 6 of 19 subjects who received at least 1 × 107/kg NK cells at cycle 1 had stable disease for ≥6 months (median, 8.8 months; range 6.0-12.0). One patient, the only one with the high-affinity F158V CD16 variant, had a partial response. Peripheral blood NK cells progressively downregulated CD16 postinfusion; paired tumor biopsies showed increased NK cells, lymphocytic infiltrates, and apoptosis posttreatment. CONCLUSIONS: NK-cell therapy in combination with trastuzumab was well tolerated, with target engagement and preliminary antitumor activity, supporting continued assessment of this approach in phase II trials.

The Role of Liver-Directed Therapy in Metastatic Colorectal Cancer.

PURPOSE OF REVIEW: Colorectal cancer liver metastasis is a major clinical problem, and surgical resection is the only potentially curative treatment. We seek to discuss various liver-directed therapy modalities and explore their roles in the evolving realm of treatment strategies for metastatic colorectal cancer. RECENT FINDINGS: Clinical outcomes for patients with colorectal cancer liver metastases have improved as more patients undergo potentially curative resection and as the armamentarium of systemic treatment and liver-directed therapies continues to expand. Liver-directed therapies have been developed as adjuncts to improve resectability, employed in the adjuvant setting to potentially reduce local recurrence rates, and utilized in the palliative setting with the aim to improve overall survival. SUMMARY: Ongoing research is expected to validate the role of these evolving therapeutic options, and determine how best to sequence and when to apply these therapies.

Prognostic significance of immune cells in non-small cell lung cancer: meta-analysis.

BACKGROUND: Tumor-associated immune cells are prognostic in non-small cell lung cancer (NSCLC) but findings have been conflicting. OBJECTIVES: To determine the prognostic role of immune cells according to localization in NSCLC patients. METHODS: A systematic literature review and meta-analysis was performed on dendritic cell (DC), tumor associated macrophages (TAM), mast cells (MC), natural killer (NK) cells, T and B cells and tumor CTLA-4 and PD-L1 studies. RESULTS: We analysed 96 articles (n= 21,752 patients). Improved outcomes were seen with increased tumor DCs (overall survival (OS) hazard ratio (HR) 0.55; 95% confidence interval (CI) 0.44-0.68), NK cells (OS HR 0.45; 0.31-0.65), TAMs (OS HR 0.33; 0.17-0.62), M1 TAMs (OS HR 0.10; 0.05-0.21), CD3+ T cells (disease specific survival (DSS) HR 0.64; 0.48-0.86), CD8+ T cells (OS HR 0.78; 0.66-0.93), B cells (OS HR 0.65; 0.42-0.99) and with increased stroma DC (DSS HR 0.62; 0.47-0.83), NK cells (DSS HR 0.51; 0.32-0.82), M1 TAMs (OS HR 0.63; 0.42-0.94), CD4+ T cells (OS HR 0.45; 0.21-0.94), CD8+ T cells (OS HR 0.77; 0.69-0.86) and B cells (OS HR 0.74;0.56-0.99). Poor outcomes were seen with stromal M2 TAMs (OS HR 1.44; 1.06-1.96) and Tregs (relapse free survival (RFS) HR 1.80; 1.34-2.43). Tumor PD-L1 was associated with worse OS (1.40; 1.20-1.69), RFS (1.67) and DFS (1.24). CONCLUSION: Tumor and stroma DC, NK cells, M1 TAMs, CD8+ T cells and B cells were associated with improved prognosis and tumor PD-L1, stromal M2 TAMs and Treg cells had poorer prognosis. Higher quality studies are required for confirmation.

Study protocol: phase 1 dose escalating study of Pressurized Intra-Peritoneal Aerosol Chemotherapy (PIPAC) with oxaliplatin in peritoneal metastasis.

BACKGROUND: Pressurized Intra-Peritoneal Aerosol Chemotherapy (PIPAC) is a novel laparoscopic intraperitoneal chemotherapy technique, with advantages such as homogeneous distribution of aerosol and deeper tissue penetration. Thus far, PIPAC oxaliplatin has been administered at an arbitrary dose of 92 mg/m2. AIM: We aim to determine the dose-related safety profile and tolerability of PIPAC oxaliplatin using an evidence-based approach. The secondary aim is to evaluate clinic-pathologic response and the pharmacokinetic profile. METHODS: This is a phase I 3+3 dose escalation study for gastric and colorectal cancer with predominant peritoneal metastasis starting at a dose of 45 mg/m2. Safety is assessed according to Clavien-Dindo Classification and Common Terminology Criteria for Adverse Events (version 4.0). Clinico-pathologic response is assessed using the Peritoneal Regression Grading Score, Peritoneal Cancer Index, and Response Evaluation Criteria In Solid Tumour criteria (version 1.1). Pharmacokinetic analysis is performed using Inductively Coupled Plasma-Mass Spectrometry assay. This trial is registered on ClinicalTrials.gov (NCT03172416). CONCLUSIONS: This phase I study can provide the scientific basis to identify the optimal dose for PIPAC with oxaliplatin such that the benefits of this novel and promising intraperitoneal chemotherapy delivery technique can be maximized.

Best practice in the treatment of advanced squamous cell lung cancer.

The management of advanced stage nonsmall cell lung cancer (NSCLC) has been altered by the recognition of histology-based treatment and the use of targeted therapy. Whilst outcomes have improved with adenocarcinoma, treatment options are still limited in advanced stage squamous cell lung cancer. With advances in the molecular characterization of squamous cell cancers (SCCs), new potential targets have been identified. In this review, we discuss the role of histology in the treatment of NSCLC, cytotoxic chemotherapy, existing targeted therapies, the new molecular subsets and novel inhibitors in squamous cell lung carcinoma, and the emerging role of immune checkpoint inhibitors. Based on the results of two recent studies, nivolumab, a programmed death 1 (PD-1) immune checkpoint inhibitor, has been approved by the US Food and Drug Administration (FDA) in the treatment of squamous cell NSCLC in the second-line setting. Well-designed biomarker driven studies are needed to accelerate the development and approval of novel therapies for patients with lung SCC.