Pembrolizumab alone and pembrolizumab plus chemotherapy in previously treated, extrapulmonary poorly differentiated neuroendocrine carcinomas.

BACKGROUND: To date, single-agent immune checkpoint inhibitor (CPI) therapy has proven to be ineffective against biomarker-unselected extrapulmonary poorly differentiated neuroendocrine carcinomas (EP-PDNECs). The efficacy of CPI in combination with chemotherapy remains under investigation. METHODS: Patients with advanced, progressive EP-PDNECs were enrolled in a two-part study of pembrolizumab-based therapy. In Part A, patients received pembrolizumab alone. In Part B, patients received pembrolizumab plus chemotherapy. PRIMARY ENDPOINT: objective response rate (ORR). Secondary endpoints: safety, progression-free survival (PFS) and overall survival (OS). Tumours were profiled for programmed death-ligand 1 expression, microsatellite-high/mismatch repair deficient status, mutational burden (TMB), genomic correlates. Tumour growth rate was evaluated. RESULTS: Part A (N = 14): ORR (pembrolizumab alone) 7% (95% CI, 0.2-33.9%), median PFS 1.8 months (95% CI, 1.7-21.4), median OS 7.8 months (95% CI, 3.1-not reached); 14% of patients (N = 2) had grade 3/4 treatment-related adverse events (TRAEs). Part B (N = 22): ORR (pembrolizumab plus chemotherapy) 5% (95% CI, 0-22.8%), median PFS 2.0 months (95% CI, 1.9-3.4), median OS 4.8 months (95% CI, 4.1-8.2); 45% of patients (N = 10) had grade 3/4 TRAEs. The two patients with objective response had high-TMB tumours. DISCUSSION: Treatment with pembrolizumab alone and pembrolizumab plus chemotherapy was ineffective in advanced, progressive EP-PDNECs. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT03136055.

Baseline tumor growth rate highlights the heterogeneity of well differentiated gastroenteropancreatic neuroendocrine tumors and predicts for increases in Ki67 index over time.

Refined risk stratification for gastroenteropancreatic neuroendocrine tumors (GEP-NETs) has the potential to improve comparisons of study populations across clinical trials and facilitate drug development. Tumor growth rate (TGR) is a radiological metric with demonstrated prognostic value in well differentiated grade 1 and 2 (G1-2) GEP-NETs, but little is known about TGR in G3 NETs. In this retrospective study of 48 patients with advanced G1-3 GEP-NET, we calculated baseline TGR (TGR0 ) from radiological images of metastases acquired prior to first-line therapy and evaluated its association with disease characteristics and outcomes. The median pretreatment Ki67 proliferation index for G1-3 tumors combined was 5% (range = 0.1%-52%) and median TGR0 was 4.8%/month (m) (range = 0%-45.9%/m). TGR0 correlated with pretreatment Ki67 across G1-3 pooled and within G3 GEP-NET. Patients with higher TGR0 (>11.7%/m) tumors, which were primarily G3 pancreatic NETs, exhibited decreased time to first therapy (median, 2.2 vs. 5.3 months; p = .03) and shorter overall survival (median, 4.1 years vs. not reached; p = .003). Independent of therapies given, higher TGR0 GEP-NETs experienced a greater incidence of Ki67 increase (100 vs. 50%; p = .02) and greater magnitude of Ki67 change (median, 14.0 vs. 0.1%; p = .04) upon serial biopsy. Importantly, TGR0 , but not grade, predicted for future Ki67 increase in this series. Given the heterogeneity of well differentiated GEP-NETs, future clinical trials may benefit from stratification for TGR0 , particularly in G1-2 tumors, in which TGR0 does not correlate with Ki67. TGR0 has the potential to noninvasively identify patients with previously undiagnosed grade progression and those in whom more or less frequent monitoring may be appropriate. Additional research is needed to determine the prognostic and predictive value of TGR0 in larger and more homogeneously treated cohorts, and to ascertain if post-treatment TGR has value in previously treated patients starting a new line of therapy.

Therapeutically reprogrammed nutrient signalling enhances nanoparticulate albumin bound drug uptake and efficacy in KRAS-mutant cancer.

Nanoparticulate albumin bound paclitaxel (nab-paclitaxel, nab-PTX) is among the most widely prescribed nanomedicines in clinical use, yet it remains unclear how nanoformulation affects nab-PTX behaviour in the tumour microenvironment. Here, we quantified the biodistribution of the albumin carrier and its chemotherapeutic payload in optically cleared tumours of genetically engineered mouse models, and compared the behaviour of nab-PTX with other clinically relevant nanoparticles. We found that nab-PTX uptake is profoundly and distinctly affected by cancer-cell autonomous RAS signalling, and RAS/RAF/MEK/ERK inhibition blocked its selective delivery and efficacy. In contrast, a targeted screen revealed that IGF1R kinase inhibitors enhance uptake and efficacy of nab-PTX by mimicking glucose deprivation and promoting macropinocytosis via AMPK, a nutrient sensor in cells. This study thus shows how nanoparticulate albumin bound drug efficacy can be therapeutically improved by reprogramming nutrient signalling and enhancing macropinocytosis in cancer cells.

Efficient blockade of locally reciprocated tumor-macrophage signaling using a TAM-avid nanotherapy.

Interpreting how multicellular interactions in the tumor affect resistance pathways to BRAF and MEK1/2 MAPK inhibitors (MAPKi) remains a challenge. To investigate this, we profiled global ligand-receptor interactions among tumor and stromal/immune cells from biopsies of MAPK-driven disease. MAPKi increased tumor-associated macrophages (TAMs) in some patients, which correlated with poor clinical response, and MAPKi coamplified bidirectional tumor-TAM signaling via receptor tyrosine kinases (RTKs) including AXL, MERTK, and their ligand GAS6. In xenograft tumors, intravital microscopy simultaneously monitored in situ single-cell activities of multiple kinases downstream of RTKs, revealing MAPKi increased TAMs and enhanced bypass signaling in TAM-proximal tumor cells. As a proof-of-principle strategy to block this signaling, we developed a multi-RTK kinase inhibitor nanoformulation that accumulated in TAMs and delayed disease progression. Thus, bypass signaling can reciprocally amplify across nearby cell types, offering new opportunities for therapeutic design.

Correction: Combined MEK and ERK inhibition overcomes therapy-mediated pathway reactivation in RAS mutant tumors.

[This corrects the article DOI: 10.1371/journal.pone.0185862.].

Combined MEK and ERK inhibition overcomes therapy-mediated pathway reactivation in RAS mutant tumors.

Mitogen-activated protein kinase (MAPK) pathway dysregulation is implicated in >30% of all cancers, rationalizing the development of RAF, MEK and ERK inhibitors. While BRAF and MEK inhibitors improve BRAF mutant melanoma patient outcomes, these inhibitors had limited success in other MAPK dysregulated tumors, with insufficient pathway suppression and likely pathway reactivation. In this study we show that inhibition of either MEK or ERK alone only transiently inhibits the MAPK pathway due to feedback reactivation. Simultaneous targeting of both MEK and ERK nodes results in deeper and more durable suppression of MAPK signaling that is not achievable with any dose of single agent, in tumors where feedback reactivation occurs. Strikingly, combined MEK and ERK inhibition is synergistic in RAS mutant models but only additive in BRAF mutant models where the RAF complex is dissociated from RAS and thus feedback productivity is disabled. We discovered that pathway reactivation in RAS mutant models occurs at the level of CRAF with combination treatment resulting in a markedly more active pool of CRAF. However, distinct from single node targeting, combining MEK and ERK inhibitor treatment effectively blocks the downstream signaling as assessed by transcriptional signatures and phospho-p90RSK. Importantly, these data reveal that MAPK pathway inhibitors whose activity is attenuated due to feedback reactivation can be rescued with sufficient inhibition by using a combination of MEK and ERK inhibitors. The MEK and ERK combination significantly suppresses MAPK pathway output and tumor growth in vivo to a greater extent than the maximum tolerated doses of single agents, and results in improved anti-tumor activity in multiple xenografts as well as in two Kras mutant genetically engineered mouse (GEM) models. Collectively, these data demonstrate that combined MEK and ERK inhibition is functionally unique, yielding greater than additive anti-tumor effects and elucidates a highly effective combination strategy in MAPK-dependent cancer, such as KRAS mutant tumors.

Reduced Proteolytic Shedding of Receptor Tyrosine Kinases Is a Post-Translational Mechanism of Kinase Inhibitor Resistance.

UNLABELLED: Kinase inhibitor resistance often involves upregulation of poorly understood "bypass" signaling pathways. Here, we show that extracellular proteomic adaptation is one path to bypass signaling and drug resistance. Proteolytic shedding of surface receptors, which can provide negative feedback on signaling activity, is blocked by kinase inhibitor treatment and enhances bypass signaling. In particular, MEK inhibition broadly decreases shedding of multiple receptor tyrosine kinases (RTK), including HER4, MET, and most prominently AXL, an ADAM10 and ADAM17 substrate, thus increasing surface RTK levels and mitogenic signaling. Progression-free survival of patients with melanoma treated with clinical BRAF/MEK inhibitors inversely correlates with RTK shedding reduction following treatment, as measured noninvasively in blood plasma. Disrupting protease inhibition by neutralizing TIMP1 improves MAPK inhibitor efficacy, and combined MAPK/AXL inhibition synergistically reduces tumor growth and metastasis in xenograft models. Altogether, extracellular proteomic rewiring through reduced RTK shedding represents a surprising mechanism for bypass signaling in cancer drug resistance. SIGNIFICANCE: Genetic, epigenetic, and gene expression alterations often fail to explain adaptive drug resistance in cancer. This work presents a novel post-translational mechanism of such resistance: Kinase inhibitors, particularly targeting MAPK signaling, increase tumor cell surface receptor levels due to widely reduced proteolysis, allowing tumor signaling to circumvent intended drug action.

Simultaneous concentration and detection of biomarkers on paper.

The lateral-flow immunoassay (LFA) is an inexpensive point-of-care (POC) paper-based diagnostic device with the potential to rapidly detect disease biomarkers in resource-poor settings. Although LFA is inexpensive, easy to use, and requires no laboratory equipment, it is limited by its sensitivity, which remains inferior to that of gold standard laboratory-based assays. Our group is the only one to have previously utilized various aqueous two-phase systems (ATPSs) to enhance LFA detection. In those studies, the sample was concentrated by an ATPS in a test tube and could only be applied to LFA after it had been extracted manually. Here, we bypass the extraction step by seamlessly integrating a polyethylene glycol-potassium phosphate ATPS with downstream LFA detection in a simple, inexpensive, power-free, and portable all-in-one diagnostic device. We discovered a new phenomenon in which the target biomarkers simultaneously concentrate as the ATPS solution flows through the paper membranes, and our device features a 3-D paper well that was designed to exploit this phenomenon. Studies with this device, which were performed at room temperature in under 25 min, demonstrated a 10-fold improvement in the detection limit of a model protein, transferrin. Our next-generation LFA technology is rapid, affordable, easy-to-use, and can be applied to existing LFA products, thereby providing a new platform for revolutionizing the current state of disease diagnosis in resource-poor settings.

Improving the systemic drug delivery efficacy of nanoparticles using a transferrin variant for targeting.

Targeted therapy for the treatment of cancers using nanoparticles (NPs) decorated with transferrin (Tf) has been relatively successful, as several such nanocarriers are currently undergoing clinical trials. However, since native Tf has a low probability of delivering its payload due to its short residence time in the cell, or low cellular association, there is room to significantly improve the potency of current systems. We pioneered the redesign of this targeting ligand by altering the ligand-metal interaction, as suggested by our mathematical model, and here we present the first study to investigate the enhanced therapeutic efficacy of NPs conjugated to our engineered oxalate Tf. Our mathematical model was first used to predict that NPs conjugated to oxalate Tf will exhibit a higher degree of cellular association compared to native Tf-conjugated NPs. Our in vitro trafficking experiments validated the model prediction, and subsequent in vitro and in vivo efficacy studies demonstrated that this increase in cellular association further translates into an enhanced ability to deliver chemotherapeutics. Our findings signify the importance of the cellular trafficking properties of targeting ligands, as they may significantly influence therapeutic potency when such ligands are conjugated to NPs. Given the early success of a number of native Tf-conjugated NPs in clinical trials, there is potential for using Tf-variant based therapeutics in systemic drug delivery applications for cancer treatment.