Crizotinib in patients with tumors harboring ALK or ROS1 rearrangements in the NCI-MATCH trial.

The NCI-MATCH was designed to characterize the efficacy of targeted therapies in histology-agnostic driver mutation-positive malignancies. Sub-protocols F and G were developed to evaluate the role of crizotinib in rare tumors that harbored either ALK or ROS1 rearrangements. Patients with malignancies that progressed following at least one prior systemic therapy were accrued to the NCI-MATCH for molecular profiling, and those with actionable ALK or ROS1 rearrangements were offered participation in sub-protocols F or G, respectively. There were five patients who enrolled on Arm F (ALK) and four patients on Arm G (ROS1). Few grade 3 or 4 toxicities were noted, including liver test abnormalities, and acute kidney injury. For sub-protocol F (ALK), the response rate was 50% (90% CI 9.8-90.2%) with one complete response among the 4 eligible patients. The median PFS was 3.8 months, and median OS was 4.3 months. For sub-protocol G (ROS1) the response rate was 25% (90% CI 1.3-75.1%). The median PFS was 4.3 months, and median OS 6.2 months. Data from 3 commercial vendors showed that the prevalence of ALK and ROS1 rearrangements in histologies other than non-small cell lung cancer and lymphoma was rare (0.1% and 0.4% respectively). We observed responses to crizotinib which met the primary endpoint for ALK fusions, albeit in a small number of patients. Despite the limited accrual, some of the patients with these oncogenic fusions can respond to crizotinib which may have a therapeutic role in this setting.

Outcome of neoadjuvant treatment for pancreatic cancer in elderly patients: comparative, observational cohort study.

BACKGROUND: Use of neoadjuvant therapy for elderly patients with pancreatic cancer has been debatable. With FOLFIRINOX (folinic acid, 5-fluorouracil, irinotecan, oxaliplatin) or gemcitabine plus nab-paclitaxel (GnP) showing tremendous effects in improving the overall survival of patients with borderline resectable and locally advanced pancreatic cancer, there is no definitive consensus regarding the use of this regimen in the elderly. METHODS: This study evaluated the eligibility of elderly patients with borderline resectable or locally advanced pancreatic cancer for neoadjuvant therapy. Patients registered in the database of pancreatic cancer at the University of Colorado Cancer Center, who underwent neoadjuvant treatment between January 2011 and March 2019, were separated into three age groups (less than 70, 70-74, 75 or more years) and respective treatment outcomes were compared. RESULTS: The study included 246 patients with pancreatic cancer who underwent neoadjuvant treatment, of whom 154 and 71 received chemotherapy with FOLFIRINOX and GnP respectively. Among these 225 patients, 155 were younger than 70 years, 36 were aged 70-74 years, and 34 were aged 75 years or older. Patients under 70 years old received FOLFIRINOX most frequently (124 of 155 versus 18 of 36 aged 70-74 years, and 12 of 34 aged 75 years or more; P < 0.001). Resectability was similar among the three groups (60.0, 58.3, and 55.9 per cent respectively; P = 0.919). Trends towards shorter survival were observed in the elderly (median overall survival time 23.6, 18.0, and 17.6 months for patients aged less than 70, 70-74, and 75 or more years respectively; P = 0.090). After adjusting for co-variables, age was not a significant predictive factor. CONCLUSION: The safety and efficacy of multiagent chemotherapy in patients aged 75 years or over were similar to those in younger patients. Modern multiagent regimens could be a safe and viable treatment option for clinically fit patients aged at least 75 years.

Characterization of immune responses to anti-PD-1 mono and combination immunotherapy in hematopoietic humanized mice implanted with tumor xenografts.

BACKGROUND: The success of agents that reverse T-cell inhibitory signals, such as anti-PD-1/PD-L1 therapies, has reinvigorated cancer immunotherapy research. However, since only a minority of patients respond to single-agent therapies, methods to test the potential anti-tumor activity of rational combination therapies are still needed. Conventional murine xenograft models have been hampered by their immune-compromised status; thus, we developed a hematopoietic humanized mouse model, hu-CB-BRGS, and used it to study anti-tumor human immune responses to triple-negative breast cancer (TNBC) cell line and patient-derived colorectal cancer (CRC) xenografts (PDX). METHODS: BALB/c-Rag2nullIl2rγnullSIRPαNOD (BRGS) pups were humanized through transplantation of cord blood (CB)-derived CD34+ cells. Mice were evaluated for human chimerism in the blood and assigned into experimental untreated or nivolumab groups based on chimerism. TNBC cell lines or tumor tissue from established CRC PDX models were implanted into both flanks of humanized mice and treatments ensued once tumors reached a volume of ~150mm3. Tumors were measured twice weekly. At end of study, immune organs and tumors were collected for immunological assessment. RESULTS: Humanized PDX models were successfully established with a high frequency of tumor engraftment. Humanized mice treated with anti-PD-1 exhibited increased anti-tumor human T-cell responses coupled with decreased Treg and myeloid populations that correlated with tumor growth inhibition. Combination therapies with anti-PD-1 treatment in TNBC-bearing mice reduced tumor growth in multi-drug cohorts. Finally, as observed in human colorectal patients, anti-PD-1 therapy had a strong response to a microsatellite-high CRC PDX that correlated with a higher number of human CD8+ IFNγ+ T cells in the tumor. CONCLUSION: Hu-CB-BRGS mice represent an in vivo model to study immune checkpoint blockade to human tumors. The human immune system in the mice is inherently suppressed, similar to a tumor microenvironment, and thus allows growth of human tumors. However, the suppression can be released by anti-PD-1 therapies and inhibit tumor growth of some tumors. The model offers ample access to lymph and tumor cells for in-depth immunological analysis. The tumor growth inhibition correlates with increased CD8 IFNγ+ tumor infiltrating T cells. These hu-CB-BRGS mice provide a relevant preclinical animal model to facilitate prioritization of hypothesis-driven combination immunotherapies.

Cytokine profile and prognostic significance of high neutrophil-lymphocyte ratio in colorectal cancer.

BACKGROUND: High circulating neutrophil-lymphocyte ratio (NLR) appears to be prognostic in metastatic colorectal cancer (mCRC). We investigated the relationship of NLR with circulating cytokines and molecular alterations. METHODS: We performed retrospective analyses on multiple cohorts of CRC patients (metastatic untreated (n=166), refractory metastatic (n=161), hepatectomy (n=198), stage 2/3 (n=274), and molecularly screened (n=342)). High NLR (ratio of absolute neutrophil-to-lymphocyte counts in peripheral blood) was defined as NLR>5. Plasma cytokines were evaluated using multiplex-bead assays. Kaplan-Meier estimates, non-parametric correlation analysis, and hierarchical cluster analyses were used. RESULTS: High NLR was associated with poor prognosis in mCRC (hazard ratio (HR) 1.73; 95% confidence interval (CI):1.03-2.89; P=0.039) independent of known prognostic factors and molecular alterations (KRAS/NRAS/BRAF/PIK3CA/CIMP). High NLR correlated with increased expression of interleukin 6 (IL-6), IL-8, IL-2Rα, hepatocyte growth factor, macrophage-colony stimulating factor, and vascular epidermal growth factor in exploratory (n=39) and validation (n=166) cohorts. Fourteen additional cytokines correlated with high NLR in the validation cohort. All 20 cytokines fell into three major clusters: inflammatory cytokines, angiogenic cytokines, and epidermal growth factor ligands. In mCRC, composite stratification based on NLR-cytokine score provided enhanced prognostic information (HR 2.09; 95% CI: 1.59-2.76; P<0.001) over and above NLR. CONCLUSIONS: High NLR is an independent poor prognostic marker in CRC and correlates with a distinct cytokine profile related to key biological processes involved in carcinogenesis. A composite NLR-cytokine stratification has enhanced prognostic value in mCRC.

Systemic chemotherapy and surgical cytoreduction for poorly differentiated and signet ring cell adenocarcinomas of the appendix.

BACKGROUND: Poorly differentiated and signet ring cell adenocarcinomas of the appendix represent a subset with aggressive tumor biology and poor outcomes with few studies evaluating the impact of systemic chemotherapy and cytoreductive surgery (CRS). PATIENTS AND METHODS: A retrospective chart review of patients with either poorly differentiated and signet ring cell appendiceal adenocarcinomas was completed from 1992 to 2010. RESULTS: One hundred forty-two patients were identified. Seventy-eight patients with metastatic disease received chemotherapy. Radiographic response was 44%, median progression-free survival (PFS) was 6.9 months, and median overall survival (OS) was 1.7 years. In multivariate analysis, response to chemotherapy [hazard ratio (HR) 0.5; P = 0.02] predicted improved PFS, and complete CRS (HR 0.3; P = 0.004) predicted improved OS. Patients who underwent complete CRS (n = 26) had a median relapse-free survival (RFS) of 1.2 years and a median OS of 4.2 years. In multivariate analysis for this subset, complete cytoreduction score of 0 was significantly correlated with improved RFS (HR 0.07; P = 0.01) and OS (HR 0.02; P = 0.01). CONCLUSIONS: Systemic chemotherapy appears to be a viable treatment option for patients with metastatic poorly differentiated and signet ring cell appendiceal adenocarcinomas. Complete CRS is associated with improved RFS and OS, though part of this benefit likely reflects the selection of good tumor biology.

Evaluation of anti-Wnt/β-catenin signaling agents by pGL4-TOP transfected stable cells with a luciferase reporter system

Refractory and relapsed leukemia is a major problem during cancer therapy, which is due to the aberrant activation of Wnt/β-catenin signaling pathway. Activation of this pathway is promoted by wingless (Wnt) proteins and induces co-activator β-catenin binding to lymphoid enhancer factor (LEF)/T-cell factor protein (TCF). To provide a convenient system for the screening of anti-Wnt/β-catenin agents, we designed a bi-functional pGL4-TOP reporter plasmid that contained 3X β-catenin/LEF/TCF binding sites and a selectable marker. After transfection and hygromycin B selection, HEK 293-TOP and Jurkat-TOP stable clones were established. The luciferase activity in the stable clone was enhanced by the recombinant Wnt-3A (rWnt-3A; 100-400 ng/mL) and GSK3β inhibitor (2’Z,3’E)-6-bromoindirubin-3’-oxime (BIO; 5 µM) but was inhibited by aspirin (5 mM). Using this reporter model, we found that norcantharidin (NCTD; 100 µM) reduced 80 percent of rWnt-3A-induced luciferase activity. Furthermore, 50 µM NCTD inhibited 38 percent of BIO-induced luciferase activity in Jurkat-TOP stable cells. Employing ³H-thymidine uptake assay and Western blot analysis, we confirmed that NCTD (50 µM) significantly inhibited proliferation of Jurkat cells by 64 percent, which are the dominant β-catenin signaling cells and decreased β-catenin protein in a concentration-dependent manner. Thus, we established a stable HEK 293-TOP clone and successfully used it to identify the Wnt/β-catenin signaling inhibitor NCTD.

Evaluation of anti-Wnt/ß-catenin signaling agents by pGL4-TOP transfected stable cells with a luciferase reporter system.

Refractory and relapsed leukemia is a major problem during cancer therapy, which is due to the aberrant activation of Wnt/ß-catenin signaling pathway. Activation of this pathway is promoted by wingless (Wnt) proteins and induces co-activator ß-catenin binding to lymphoid enhancer factor (LEF)/T-cell factor protein (TCF). To provide a convenient system for the screening of anti-Wnt/ß-catenin agents, we designed a bi-functional pGL4-TOP reporter plasmid that contained 3X ß-catenin/LEF/TCF binding sites and a selectable marker. After transfection and hygromycin B selection, HEK 293-TOP and Jurkat-TOP stable clones were established. The luciferase activity in the stable clone was enhanced by the recombinant Wnt-3A (rWnt-3A; 100-400 ng/mL) and GSK3ß inhibitor (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO; 5 µM) but was inhibited by aspirin (5 mM). Using this reporter model, we found that norcantharidin (NCTD; 100 µM) reduced 80% of rWnt-3A-induced luciferase activity. Furthermore, 50 µM NCTD inhibited 38% of BIO-induced luciferase activity in Jurkat-TOP stable cells. Employing ³H-thymidine uptake assay and Western blot analysis, we confirmed that NCTD (50 µM) significantly inhibited proliferation of Jurkat cells by 64%, which are the dominant ß-catenin signaling cells and decreased ß-catenin protein in a concentration-dependent manner. Thus, we established a stable HEK 293-TOP clone and successfully used it to identify the Wnt/ß-catenin signaling inhibitor NCTD.

A dose-ranging study of the pharmacokinetics and pharmacodynamics of the selective apoptotic antineoplastic drug (SAAND), OSI-461, in patients with advanced cancer, in the fasted and fed state.

PURPOSE: To evaluate the safety, pharmacokinetics and determine the recommended dose of the selective apoptotic antineoplastic drug, OSI-461 administered on a twice-daily regimen to patients with advanced solid malignancies. METHODS: In this phase I trial, 33 patients were treated with OSI-461 doses ranging from 400 to 1,200 mg given twice daily in 4-week cycles. Pharmacokinetic studies were performed to characterize the plasma disposition of OSI-461 and the effect of food intake on OSI-461 absorption. Secondary biomarker studies were performed to assess the biologic activity of OSI-461 including the measurement of pGSK-3beta, a PKG substrate, and pharmacogenetic studies to identify polymorphisms of CYP3A that influence drug metabolism and of ABCG2, involved in drug resistance. RESULTS: Thirty-three patients were treated with 86 courses of OSI-461. The dose-limiting toxicities were grade 3 abdominal pain, found in one patient at the 1,000 mg BID fed dose level and all patients at the 1,200 mg BID fed dose level. There was also one episode each of grade 3 fatigue and grade 3 constipation at the 1,000 and 1,200 mg BID fed dose levels, respectively. Other common toxicities included mild to moderate fatigue, nausea, anorexia and mild elevation in bilirubin. Pharmacokinetic studies of OSI-461 revealed approximately a twofold increase in AUC(0-24) when OSI-461 was administered with food. An increase in pGSK-3beta post-dose was seen in the majority of patients and was greater at higher dose levels. No patients exhibited CYP3A4 polymorphisms, while 100% of patients were found to have the CYP3A5*3/CYP3A5*3 polymorphism. Two known polymorphisms of the ABCG2 gene, G34 --> A34 and C421 --> A421, occurred at frequencies of 11.76 and 29%, respectively. CONCLUSIONS: Toxicity and pharmacodynamic data show that the recommended oral dose of OSI-461 is 800 mg twice daily administered with food. The drug appears to be well-tolerated, and overall bioavailability appears to be markedly increased when the drug is administered with food. These results support further disease-directed evaluations of OSI-461 at a dose of 800 mg BID in combination with other chemotherapeutic agents.

Prevalence of the JAK2-V617F mutation in Taiwanese patients with chronic myeloproliferative disorders.

BACKGROUND: The Janus kinase-2 (JAK-2) V617F mutation has been recently reported in patients with myeloproliferative disorders (MPD), which is believed to underlie growth factor hypersensitivity displayed by haematopoietic progenitors in these disorders. However, its frequency has been rarely determined in Taiwanese patients. METHODS: The frequency of JAK2-V617F mutation in patients with polycythaemia vera, essential thrombocythaemia and idiopathic myelofibrosis (IMF) was determined in the DNA from the peripheral blood leucocytes of 108 patients by genomic polymerase chain reaction and restriction enzyme-based assay. RESULTS: The JAK2-V617F mutation could be detected in 28 of 33 polycythaemia vera patients (85%), 29 of 49 essential thrombocythaemia patients (59%) and 2 of 6 IMF patients (33%), but was not detected in 11 patients with myelodysplastic syndrome or another 10 with other haematological diseases. The presence of the JAK2 mutation was associated with specific MPD disease subtypes (P = 0.007), longer disease duration (P = 0.005), splenomegaly (P = 0.019), a higher white blood cell count (P = 0.002) and a higher haemoglobin level (P = 0.036). However, the overall risk of thrombosis or bleeding was not affected by the presence of the JAK2 mutation (32 vs 17%; P = 0.22). CONCLUSION: The JAK2-V617F mutation can be frequently detected in the Taiwanese patients with MPD disorders and therefore should be incorporated into the initial evaluation of patients suspected of MPD.

Role of mitogen-activated protein kinase in taxol-induced apoptosis in human leukemic U937 cells.

The induction of apoptosis by Taxol was investigated in human leukemic U937 cells. Treatment of U937 cells with 20 nM Taxol for 24 h induced apoptosis in 30-40% of cells, which resulted in an 80% growth inhibition 3 days after treatment. Synchronous cells at different cell cycle stages exhibited different sensitivities toward Taxol, and their reversion by certain protein kinase inhibitors was also phase specific. Kinetic studies of cell cycle progress reveal that Taxol accelerates the progression of the cell cycle, which facilitates the process of apoptosis, especially for cells initially in the G1 phase. This acceleration may result from transient activation of p42/ 44 mitogen-activated protein (MAP) kinase, because inhibition of upstream MAP/extracellular signal-regulated kinase kinase (MEK1/2) by PD98059 reversed this effect. However, the delayed S-G2-M-phase progression by PD98059 was insignificant. The results suggest that MAP kinase may not only mediate cell cycle progress but may also participate in the apoptosis pathway for cells originally in S phase.

Involvement of p38 mitogen-activated protein kinase signaling pathway in the rapid induction of the 78-kDa glucose-regulated protein in 9L rat brain tumor cells.

We have previously shown that treatment with okadaic acid (OA) followed by heat shock (HS) (termed OA --> HS treatment) leads to rapid transactivation of the 78-kDa glucose-regulated protein gene (grp78) in 9L rat brain tumor cells. A cAMP-responsive element-like (CRE-like, TGACGTGA) promoter sequence and a protein kinase A signaling pathway are involved in this induction, and activation of both CRE binding protein (CREB) and activating transcription factor-2 (ATF-2) is required in the above process. Herein, we report that transactivation of grp78, as well as phosphorylation/activation of ATF-2, can be completely annihilated by SB203580, a highly specific inhibitor of p38 mitogen-activated protein kinase (p38(MAPK)). Activation of p38(MAPK) by OA --> HS is also substantiated by its own phosphorylation as well as the phosphorylation and activation of MAPK activating protein kinase-2 in cells subjected to this treatment. The involvement of p38(MAPK) in the activation of ATF-2, which leads to the transactivation of rat grp78, is confirmed by electrophoretic mobility shift assay using a probe containing the CRE-like sequence as well as by transient transfection assays with a plasmid containing a 710-base pair stretch of the grp78 promoter. Together with our previous studies, these results led us to conclude that phosphorylation/activation of CREB upon OA --> HS treatment is mediated by cAMP-dependent protein kinase, whereas that of ATF-2 is mediated by p38(MAPK). The transcription factors may bind to each other to form heterodimers that in turn transactivate grp78 by binding to the CRE-like element. This suggests that distinct signaling pathways converge on CREB-ATF-2, where each subunit is individually activated by a specific class of protein kinases. This may allow modulation of grp78 transactivation by diverse external stimuli.

Dual cytotoxic mechanisms of submicromolar taxol on human leukemia HL-60 cells.

Taxol-induced mitotic block and apoptosis were investigated using taxol-sensitive human leukemia HL-60 cells at submicromolar concentrations of the drug. Cells exposed to either 20 nM taxol for 1 hr or 10 nM taxol for 12 hr were able to resume normal growth, whereas cells exposed to 60 nM taxol for 1 hr or 10 nM taxol for 24 hr failed to proliferate after drug removal. Progressive changes in the percentage of mitotic block and apoptosis induced by these four treatment protocols were monitored continuously for 3-5 days after drug removal. Cells treated with 20 nM taxol for 1 hr showed a mitotic block without a subsequent increase in apoptosis, whereas cells treated with 10 nM taxol for 12 hr showed an increase in apoptotic ratio within several hours without an increase in mitotic block. These results indicate that apoptosis does not necessarily result from mitotic block and that these two phenomena can occur independently of each other. Drug sensitivity at progressive stages of the cell cycle was also investigated. The results showed that, in addition to the cells in G2/M phase, the cells in S phase were also sensitive to the drug, especially to a prolonged treatment. These results suggest that, in HL-60 cells, the apoptotic programs can be initiated in either the G2/M or S phase and represent two different cytotoxic mechanisms of taxol.