Microscopy imaging of mouse growth plates is extensively used in biology to understand the effect of specific molecules on various stages of normal bone development and on bone disease. Until now, such image analysis has been conducted by manual detection. In fact, when existing automated detection techniques were applied, morphological variations across the growth plate and heterogeneity of image background color, including the faint presence of cells (chondrocytes) located deeper in tissue away from the image's plane of focus, and lack of cell-specific features, interfered with identification of cell. We propose the first method of automated detection and morphometry applicable to images of cells in the growth plate of long bone. Through ad hoc sequential application of the Retinex method, anisotropic diffusion and thresholding, our new cell detection algorithm (CDA) addresses these challenges on bright-field microscopy images of mouse growth plates. Five parameters, chosen by the user in respect of image characteristics, regulate our CDA. Our results demonstrate effectiveness of the proposed numerical method relative to manual methods. Our CDA confirms previously established results regarding chondrocytes' number, area, orientation, height and shape of normal growth plates. Our CDA also confirms differences previously found between the genetic mutated mouse Smad1/5^CKO and its control mouse on fluorescence images. The CDA aims to aid biomedical research by increasing efficiency and consistency of data collection regarding arrangement and characteristics of chondrocytes. Our results suggest that automated extraction of data from microscopy imaging of growth plates can assist in unlocking information on normal and pathological development, key to the underlying biological mechanisms of bone growth.

Background

Preclinical data suggest that vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β signaling interact to stimulate angiogenesis and suppress antitumor immune responses. Thus, combined inhibition of both pathways may offer greater antitumor activity compared with VEGF-targeted antiangiogenic monotherapy against hepatocellular carcinoma (HCC).

Methods

This is a multicenter, open-label, phase 1b study of galunisertib, an inhibitor of TGF-β receptor 1, and ramucirumab, an anti-VEGF receptor 2 antibody, in patients with advanced HCC aiming to define the maximum tolerated dose (MTD). Secondary objectives included safety, pharmacokinetics (PK), antitumor efficacy, and plasma alpha-fetoprotein and TGF-β kinetics. Dose escalation employed a 3 + 3 design. Patients received galunisertib at 80 mg (cohort 1) or 150 mg (cohort 2) orally twice a day on days 1-14 of a 28-day cycle combined with ramucirumab 8 mg/kg intravenously every 2 weeks.

Results

Eight patients were enrolled: three in cohort 1 and five in cohort 2 (two patients were unevaluable due to rapid disease progression and replaced). No dose-limiting toxicities were observed. Treatment-related adverse events (AEs) of any grade in ≥2 patients included nausea (25%) and vomiting (25%). There was one Grade 3 treatment-related AE, a cerebrovascular accident possibly related to ramucirumab. Galunisertib exposure was dose-proportional and not affected by ramucirumab. The RECIST version 1.1 objective response rate and disease control rate were 0% and 12.5%, respectively.

Conclusion

Combination therapy was safe and tolerable and displayed favorable PK. The MTD was established at galunisertib at 150 mg orally twice a day and ramucirumab 8 mg/kg intravenously every 2 weeks. The results do not support the preclinical hypothesis that blocking TGFβ signaling enhances efficacy of VEGF-targeted therapy; thus further clinical development was halted for the combination of galunisertib and ramucirumab.

Lessons learned

Androgen receptor as assessed by immunohistochemistry is expressed in a high proportion of patients with hepatocellular carcinoma (HCC). Enzalutamide at 160 mg orally daily is safe and tolerable in patients with advanced HCC but has no single-agent antitumor activity. Enzalutamide, a CYP3A4 inducer, at a standard dose of 160 mg reduces the exposure of sorafenib, a CYP3A4 substrate. Enzalutamide and sorafenib is safe and tolerable in patients with advanced HCC, but the addition of enzalutamide to sorafenib did not enhance the antitumor activity of sorafenib.

Background

Androgen receptor (AR) interference is deleterious to hepatocellular carcinoma (HCC) in preclinical models.

Methods

This is a multicenter, phase Ib study of enzalutamide ± sorafenib in patients with advanced HCC. In part 1, a 3 + 3 dose de-escalation design with expansion established the recommended phase II dose (RP2D) of enzalutamide in patients in whom sorafenib treatment had failed. In part 2, a 3 + 3 dose escalation with expansion established the safety of enzalutamide with sorafenib in treatment-naive patients with HCC. Secondary objectives included objective response rate (ORR), progression-free survival (PFS), overall survival (OS), pharmacokinetics (PK), and determination of AR expression by immunohistochemistry. A 7-day run-in with sorafenib alone in part 2 allowed assessment of the impact of enzalutamide on sorafenib pharmacokinetics.

Results

In part 1, 16 patients received enzalutamide 160 mg daily. No dose-limiting toxicity (DLT) occurred; 1 patient required dose reduction. Responses were not observed; median PFS and OS were 1.8 (95% confidence interval [CI]: 1.6-3.6) and 7 (95% CI: 3.6 to not reached [NR]) months, respectively. In part 2, patients received sorafenib 400 mg daily (4) or twice a day (8) both with enzalutamide at the recommended phase II dose-no DLTs were observed. ORR was 10% (95% CI: 0.3-44.5), and median PFS and OS were 2.9 (95% CI: 1.6 to NR) and 6.7 (95% CI: 4.6 to NR) months, respectively. Enzalutamide reduced sorafenib exposure by 60%. Tumor AR expression did not associate with outcome.

Conclusion

Enzalutamide is ineffective in HCC; further development is not supported by this study.

Background

Although gemcitabine plus platinum chemotherapy is the established first-line regimen for advanced biliary cancer (ABC), there is no standard second-line therapy. This study evaluated current practice and outcomes for second-line chemotherapy in patients with ABC across 3 US academic medical centers.

Methods

Institutional registries were reviewed to identify patients who had received second-line chemotherapy for ABC from April 2010 to March 2015 along with their demographics, diagnoses and staging, treatment histories, and clinical outcomes. Overall survival from the initiation of second-line chemotherapy (OS2) was estimated with Kaplan-Meier methods.

Results

This study identified 198 patients with cholangiocarcinoma (intrahepatic [61.1%] or extrahepatic [14.1%]) or gallbladder carcinoma (24.8%); 52% received at least 3 lines of systemic chemotherapy. The median OS2 was 11 months (95% confidence interval [CI], 8.8-13.1 months). The median OS2 for patients with intrahepatic cholangiocarcinoma was 13.4 months (95% CI, 10.7-17.8 months), which was longer than that for patients with extrahepatic cholangiocarcinoma (6.8 months; 95% CI, 5-10.6 months) or gallbladder carcinoma (9.4 months; 95% CI, 7.2-12.3 months; P = .018). The median time to second-line treatment failure was 2.2 months (95% CI, 1.8-2.7 months), and it was similar across tumor locations (P = .60).

Conclusions

In this large cohort of patients with ABC treated across 3 academic medical centers after the failure of first-line chemotherapy, the time to treatment failure on standard therapies was short, although the median OS2 was longer than has been reported previously, and more than half of the patients received additional lines of treatment. This multicenter collaboration represents the largest cohort studied to date of second-line chemotherapy for ABC and provides a contemporary benchmark for future clinical trials.

PURPOSE: Praluzatamab ravtansine (CX-2009) is a conditionally activated Probody drug conjugate (PDC) comprising an anti-CD166 mAb conjugated to DM4, with a protease-cleavable linker and a peptide mask that limits target engagement in normal tissue and circulation. The tumor microenvironment is enriched for proteases capable of cleaving the linker, thereby releasing the mask, allowing for localized binding of CX-2009 to CD166. CX-2009 was evaluated in a phase I/II clinical trial for patients with advanced solid tumors. PATIENTS AND METHODS: Eligible patients had metastatic cancer receiving ≥2 prior treatments. CX-2009 was administered at escalating doses every 3 weeks (0.25-10 mg/kg) or every 2 weeks (4-6 mg/kg). Primary objective was to determine the safety profile and recommended phase II dose (RP2D). RESULTS: Of 99 patients enrolled, the most prevalent subtype was breast cancer (n = 45). Median number of prior therapies was 5 (range, 1-19). Dose-limiting toxicities were observed at 8 mg/kg every 3 weeks and 6 mg/kg every 2 weeks. On the basis of tolerability, the RP2D was 7 mg/kg every 3 weeks. Tumor regressions were observed at doses ≥4 mg/kg. In the hormone receptor-positive/HER2-nonamplified breast cancer subset (n = 22), 2 patients (9%) had confirmed partial responses, and 10 patients (45%) had stable disease. Imaging with zirconium-labeled CX-2009 confirmed uptake in tumor lesions and shielding of major organs. Activated, unmasked CX-2009 was measurable in 18 of 22 posttreatment biopsies. CONCLUSIONS: CD166 is a novel, ubiquitously expressed target. CX-2009 is the first conditionally activated antibody-drug conjugate to CD166 to demonstrate both translational and clinical activity in a variety of tumor types.

ATP-competitive fibroblast growth factor receptor (FGFR) kinase inhibitors, including BGJ398 and Debio 1347, show antitumor activity in patients with intrahepatic cholangiocarcinoma (ICC) harboring activating FGFR2 gene fusions. Unfortunately, acquired resistance develops and is often associated with the emergence of secondary FGFR2 kinase domain mutations. Here, we report that the irreversible pan-FGFR inhibitor TAS-120 demonstrated efficacy in 4 patients with FGFR2 fusion-positive ICC who developed resistance to BGJ398 or Debio 1347. Examination of serial biopsies, circulating tumor DNA (ctDNA), and patient-derived ICC cells revealed that TAS-120 was active against multiple FGFR2 mutations conferring resistance to BGJ398 or Debio 1347. Functional assessment and modeling the clonal outgrowth of individual resistance mutations from polyclonal cell pools mirrored the resistance profiles observed clinically for each inhibitor. Our findings suggest that strategic sequencing of FGFR inhibitors, guided by serial biopsy and ctDNA analysis, may prolong the duration of benefit from FGFR inhibition in patients with FGFR2 fusion-positive ICC. SIGNIFICANCE: ATP-competitive FGFR inhibitors (BGJ398, Debio 1347) show efficacy in FGFR2-altered ICC; however, acquired FGFR2 kinase domain mutations cause drug resistance and tumor progression. We demonstrate that the irreversible FGFR inhibitor TAS-120 provides clinical benefit in patients with resistance to BGJ398 or Debio 1347 and overcomes several FGFR2 mutations in ICC models.This article is highlighted in the In This Issue feature, p. 983.