Implementing flowDensity for Automated Analysis of Bone Marrow Lymphocyte Population.

INTRODUCTION: Manual gating of flow cytometry (FCM) data for marrow cell analysis is a standard approach in current practice, although it is time- and labor-consuming. Recent advances in cytometry technology have led to significant efforts in developing partially or fully automated analysis methods. Although multiple supervised and unsupervised FCM data analysis algorithms have been developed, they have not been widely adopted by the clinical and research laboratories. In this study, we evaluated flowDensity, an open source freely available algorithm, as an automated analysis tool for classification of lymphocyte subsets in the bone marrow biopsy specimens. MATERIALS AND METHODS: FlowDensity-based gating was applied to 102 normal bone marrow samples and compared with the manual analysis. Independent expression of each cell marker was assessed for comprehensive expression analysis and visualization. RESULTS: Our findings showed a correlation between the manual and flowDensity-based gating in the lymphocyte subsets. However, flowDensity-based gating in the populations with a small number of cells in each cluster showed a low degree of correlation. Comprehensive expression analysis successfully identified and visualized the lymphocyte subsets. DISCUSSION: Our study found that although flowDensity might be a promising method for FCM data analysis, more optimization is required before implementing this algorithm into day-to-day workflow.

IGF-1R/mTOR Targeted Therapy for Ewing Sarcoma: A Meta-Analysis of Five IGF-1R-Related Trials Matched to Proteomic and Radiologic Predictive Biomarkers.

Background : Ten to fourteen percent of Ewing sarcoma (ES) study participants treated nationwide with IGF-1 receptor (IGF-1R)-targeted antibodies achieved tumor regression. Despite this success, low response rates and short response durations (approximately 7-weeks) have slowed the development of this therapy. Methods: We performed a meta-analysis of five phase-1b/2 ES-oriented trials that evaluated the anticancer activity of IGF-1R antibodies +/- mTOR inhibitors (mTORi). Our meta-analysis provided a head-to-head comparison of the clinical benefits of IGF-1R antibodies vs. the IGF-1R/mTOR-targeted combination. Available pretreatment clinical samples were semi-quantitatively scored using immunohistochemistry to detect proteins in the IGF-1R/PI3K/AKT/mTOR pathway linked to clinical response. Early PET/CT imaging, obtained within the first 2 weeks (median 10 days), were examined to determine if reduced FDG avidity was predictive of progression-free survival (PFS). Results: Among 56 ES patients treated at MD Anderson Cancer Center (MDACC) with IGF-1R antibodies, our analysis revealed a significant ~two-fold improvement in PFS that favored a combination of IGF-1R/mTORi therapy (1.6 vs. 3.3-months, p = 0.042). Low pIGF-1R in the pretreatment specimens was associated with treatment response. Reduced total-lesion glycolysis more accurately predicted the IGF-1R response than other previously reported radiological biomarkers. Conclusion: Synergistic drug combinations, and newly identified proteomic or radiological biomarkers of IGF-1R response, may be incorporated into future IGF-1R-related trials to improve the response rate in ES patients.

First-in-Man Phase I Trial of the Selective MET Inhibitor Tepotinib in Patients with Advanced Solid Tumors.

PURPOSE: Tepotinib is an oral, potent, highly selective MET inhibitor. This first-in-man phase I trial investigated the MTD of tepotinib to determine the recommended phase II dose (RP2D). PATIENTS AND METHODS: Patients received tepotinib orally according to one of three dose escalation regimens (R) on a 21-day cycle: R1, 30-400 mg once daily for 14 days; R2, 30-315 mg once daily 3 times/week; or R3, 300-1,400 mg once daily. After two cycles, treatment could continue in patients with stable disease until disease progression or unacceptable toxicity. The primary endpoint was incidence of dose-limiting toxicity (DLT) and treatment-emergent adverse events (TEAE). Secondary endpoints included safety, tolerability, pharmacokinetics, pharmacodynamics, and antitumor effects. RESULTS: One hundred and forty-nine patients received tepotinib (R1: n = 42; R2: n = 45; R3: n = 62). Although six patients reported DLTs [one patient in R1 (115 mg), three patients in R2 (60, 100, 130 mg), two patients in R3 (1,000, 1,400 mg)], the MTD was not reached at the highest tested dose of 1,400 mg daily. The RP2D of tepotinib was established as 500 mg once daily, supported by translational modeling data as sufficient to achieve ≥95% MET inhibition in ≥90% of patients. Treatment-related TEAEs were mostly grade 1 or 2 fatigue, peripheral edema, decreased appetite, nausea, vomiting, and lipase increase. The best overall response in R3 was partial response in two patients, both with MET overexpression. CONCLUSIONS: Tepotinib was well tolerated with clinical activity in MET-dysregulated tumors. The RP2D of tepotinib was established as 500 mg once daily. MET abnormalities can drive tumorigenesis. This first-in-man trial demonstrated that the potent, highly selective MET inhibitor tepotinib can reduce or stabilize tumor burden and is well tolerated at doses up to 1,400 mg once daily. An RP2D of 500 mg once daily, as determined from translational modeling and simulation integrating human population pharmacokinetic and pharmacodynamic data in tumor biopsies, is being used in ongoing clinical trials.

Dual inhibition of IGF-IR and ALK as an effective strategy to eradicate NPM-ALK+ T-cell lymphoma.

BACKGROUND: Nucleophosmin-anaplastic lymphoma kinase-expressing (NPM-ALK+) T cell lymphoma is an aggressive neoplasm. NPM-ALK, an oncogenic tyrosine kinase, plays a critical role in this lymphoma. Recently, selective ALK inhibitors have emerged as a first-line therapy for this neoplasm. Unfortunately, ALK inhibitors were hindered by emergence of resistance and relapse. We have previously demonstrated that type I insulin-like growth factor receptor (IGF-IR) is commonly expressed and activated in this lymphoma. In addition, IGF-IR and NPM-ALK are physically associated and reciprocally enhance their phosphorylation/activation. Herein, we tested the hypothesis that combined inhibition of IGF-IR and NPM-ALK could significantly improve the effects of inhibiting each kinase alone. METHODS: We used clinically utilized inhibitors of IGF-IR (picropodophyllin; PPP) and ALK (ASP3026) to assess the in vitro cellular effects of combined treatment versus treatment using a single agent. Moreover, we used a systemic NPM-ALK+ T cell lymphoma mouse model to analyze the in vivo effects of PPP and ASP3026 alone or in combination. RESULTS: Our data show that combined treatment with PPP and ASP3026 decreased the viability, proliferation, and anchorage-independent colony formation, and increased apoptosis of NPM-ALK+ T cell lymphoma cells in vitro. The in vitro effects of combined treatment were synergistic and significantly more pronounced than the effects of PPP or ASP3026 alone. Biochemically, simultaneous antagonism of IGF-IR and ALK induced more pronounced decrease in pIGF-IRY1135/1136, pNPM-ALKY646, and pSTAT3Y705 levels than antagonizing IGF-IR or ALK alone. Moreover, combined targeting of IGF-IR and NPM-ALK decreased significantly systemic lymphoma tumor growth and improved mice survival in vivo. Consistent with the in vitro results, the in vivo effects of the combined therapy were more pronounced than the effects of targeting IGF-IR or ALK alone. CONCLUSIONS: Combined targeting of IGF-IR and ALK is more effective than targeting IGF-IR or ALK alone in NPM-ALK+ T cell lymphoma. This strategy might also limit emergence of resistance to high doses of ALK inhibitors. Therefore, it could represent a successful therapeutic approach to eradicate this aggressive lymphoma. Importantly, combined inhibition is feasible because of the clinical availability of IGF-IR and ALK inhibitors. Our findings are applicable to other types of cancer where IGF-IR and ALK are simultaneously expressed.

Validation of a Preclinical Model of Diethylnitrosamine-Induced Hepatic Neoplasia in Yucatan Miniature Pigs.

OBJECTIVE: The purpose of this study was to reduce the time to tumor onset in a diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) swine model via partial liver embolization (PLE) and to characterize the model for use in translational research. METHODS: Eight Yucatan miniature pigs were injected intraperitoneally with either saline (n = 2) or DEN (n = 6) solution weekly for 12 weeks. Three of the DEN-treated pigs underwent PLE. The animals underwent periodic radiological evaluation, liver biopsy, and blood sampling, and full necropsy was performed at study termination (∼29 months). RESULTS: All DEN-treated pigs developed hepatic adenoma and HCC. PLE accelerated the time to adenoma development but not to HCC development. Biomarker analysis results showed that IGF1 levels decreased in all DEN-treated pigs as functional liver capacity decreased with progression of HCC. VEGF and IL-6 levels were positively correlated with disease progression. Immunohistochemical probing of HCC tissues demonstrated the expression of several important survival-promoting proteins. CONCLUSION: To our knowledge, we are the first to demonstrate an accelerated development of hepatic neoplasia in Yucatan miniature pigs. Our HCC swine model closely mimics the human condition (i.e., progressive disease stages and expression of relevant molecular markers) and is a viable translational model.

Type I insulin-like growth factor as a liver reserve assessment tool in hepatocellular carcinoma.

Chronic liver diseases (CLDs) encompass a wide range of illnesses, including nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and viral hepatitis. Deterioration of liver capacity, with subsequent progression into cirrhosis and hepatocellular carcinoma (HCC), ultimately leads to a further decrease in the hepatic reserve. The Child-Turcotte-Pugh scoring system is the standard tool for assessing underlying liver reserve capacity in routine practice and in clinical trials of CLD and HCC. In this review, we highlight the clinical significance of insulin-like growth factor-I (IGF-I) and the growth hormone (GH) signaling pathway in HCC. IGF-I could be a marker for liver reserve capacity in CLDs and HCC in clinical practice. This approach could improve the risk assessment and stratifications of patients on the basis of their underlying liver reserve, either before active treatment in routine practice or before they are enrolled in clinical trials.