Therapies after first-line afatinib in patients with EGFRm+ NSCLC in Japan: retrospective analysis of LUX-Lung 3.

Aim: Acquired resistance to EGFR tyrosine kinase inhibitors is inevitable in non-small-cell lung cancer. To inform subsequent treatment decisions, we retrospectively assessed therapies following afatinib in Japanese patients from LUX-Lung 3. Patients & methods: LUX-Lung 3 was a randomized, open-label, Phase III study of afatinib versus cisplatin/pemetrexed in treatment-naive patients with EGFR mutation-positive (EGFRm+) advanced lung adenocarcinoma. Results: Among 47 Japanese patients who discontinued first-line afatinib, 91/81/62% received ≥one/two/three subsequent therapies. The most common second-line therapies were platinum-based chemotherapy (38%) and a first-generation EGFR tyrosine kinase inhibitor (17%). Median overall survival (afatinib vs cisplatin/pemetrexed) was 47.8 versus 35.0 months (not significant). Conclusion: First-line afatinib does not appear to diminish suitability for subsequent therapies in EGFRm+ non-small-cell lung cancer.

Real-world treatment of over 1600 Japanese patients with EGFR mutation-positive non-small cell lung cancer with daily afatinib.

BACKGROUND: This prospective, post-marketing observational study in Japanese patients aimed to evaluate the safety and effectiveness of daily afatinib use in general practice. METHODS: This non-interventional study (NCT02131259) enrolled treatment-naïve and pre-treated patients with inoperable/recurrent EGFR mutation-positive NSCLC, eligible for afatinib treatment as per the afatinib label in Japan. Patients received afatinib at the approved dose (20, 30, 40, or 50 mg/day; physician decision), and were observed following treatment initiation for 52 weeks or until premature discontinuation. Primary endpoint was the incidence of adverse drug reactions (ADRs). Secondary endpoints included ADRs of special interest, and objective response rate (ORR). Post hoc Cox multivariate analyses were used to assess prognostic factors associated with the incidence of ADRs. RESULTS: 1602 patients, at 374 sites (April 2014-March 2015), were included in the analysis; 307 (19%) were aged ≥ 75 years. The most frequently reported ADRs (all/grade 3-4) were diarrhea (78%/15%), rash/acne (59%/6%), stomatitis (31%/4%), and nail effects (38%/4%). Serious ADRs resulting in death occurred in 18 patients (1%). 762 patients (48%) had ≥ 1 afatinib dose reduction and 366 (23%) discontinued due to ADRs; the most common reason for both was diarrhea (8.2% and 6.7%, respectively). ORR was 40.1%. CONCLUSIONS: Real-world treatment of 1602 Japanese patients with afatinib was associated with a predictable ADR profile. Afatinib showed effectiveness in inoperable/recurrent EGFR mutation-positive NSCLC, especially as first-line treatment. As with other EGFR TKIs, prompt management of adverse events is needed in the Japanese population, to reduce serious events and outcomes, including interstitial lung disease.

Correction to: Real-world treatment of over 1600 Japanese patients with EGFR mutation-positive non-small cell lung cancer with daily afatinib.

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Therapeutic liver reconstitution with murine cells isolated long after death.

BACKGROUND & AIMS: Due to the shortage of donor organs, many patients needing liver transplantation cannot receive one. For some liver diseases, hepatocyte transplantation could be a viable alternative, but donor cells currently are procured from the same sources as whole organs, and thus the supply is severely limited. METHODS: Here, we investigated the possibility of isolating viable hepatocytes for liver cell therapy from the plentiful source of morgue cadavers. To determine the utility of this approach, cells were isolated from the livers of non-heart-beating cadaveric mice long after death and transplanted into fumarylacetoacetate hydrolase-deficient mice, a model for the human metabolic liver disease hereditary tyrosinemia type I and a stringent in vivo model for hepatic cell transplantation. RESULTS: Surprisingly, complete and therapeutic liver repopulation could be achieved with hepatocytes derived up to 27 hours post mortem. CONCLUSIONS: Competitive repopulation experiments showed that cadaveric liver cells had a repopulation capacity similar to freshly isolated hepatocytes. Importantly, viable hepatocytes also could be isolated from cadaveric primate liver (monkey and human) efficiently. These data provide evidence that non-heart-beating donors could be a suitable source of hepatocytes for much longer time periods than previously thought possible.

Robust expansion of human hepatocytes in Fah-/-/Rag2-/-/Il2rg-/- mice.

Mice that could be highly repopulated with human hepatocytes would have many potential uses in drug development and research applications. The best available model of liver humanization, the uroplasminogen-activator transgenic model, has major practical limitations. To provide a broadly useful hepatic xenorepopulation system, we generated severely immunodeficient, fumarylacetoacetate hydrolase (Fah)-deficient mice. After pretreatment with a urokinase-expressing adenovirus, these animals could be highly engrafted (up to 90%) with human hepatocytes from multiple sources, including liver biopsies. Furthermore, human cells could be serially transplanted from primary donors and repopulate the liver for at least four sequential rounds. The expanded cells displayed typical human drug metabolism. This system provides a robust platform to produce high-quality human hepatocytes for tissue culture. It may also be useful for testing the toxicity of drug metabolites and for evaluating pathogens dependent on human liver cells for replication.

Gene therapy of metachromatic leukodystrophy reverses neurological damage and deficits in mice.

Metachromatic leukodystrophy (MLD) is a demyelinating lysosomal storage disorder for which new treatments are urgently needed. We previously showed that transplantation of gene-corrected hematopoietic stem progenitor cells (HSPCs) in presymptomatic myeloablated MLD mice prevented disease manifestations. Here we show that HSC gene therapy can reverse neurological deficits and neuropathological damage in affected mice, thus correcting an overt neurological disease. The efficacy of gene therapy was dependent on and proportional to arylsulfatase A (ARSA) overexpression in the microglia progeny of transplanted HSPCs. We demonstrate a widespread enzyme distribution from these cells through the CNS and a robust cross-correction of neurons and glia in vivo. Conversely, a peripheral source of enzyme, established by transplanting ARSA-overexpressing hepatocytes from transgenic donors, failed to effectively deliver the enzyme to the CNS. These results indicate that the recruitment of gene-modified, enzyme-overexpressing microglia makes the enzyme bioavailable to the brain and makes therapeutic efficacy and disease correction attainable. Overall, our data provide a strong rationale for implementing HSPC gene therapy in MLD patients.

Purification of adult hepatic progenitor cells using green fluorescent protein (GFP)-transgenic mice and fluorescence-activated cell sorting.

BACKGROUND/AIMS: Recent advances in stem cell research have revealed that hepatic stem/progenitor cells may play an important role in liver development and regeneration. However, a lack of detectable definitive markers in viable cells has hindered their primary culture from adult livers. METHODS: Enzymatically dissociated liver cells from green fluorescent protein (GFP)-transgenic mice, which express GFP highly in liver endodermal cells, were sorted by GFP expression using a fluorescence-activated cell sorter. Sorted cells were characterized, and also low-density cultured for extended periods to determine their proliferation and clonal differentiation capacities. RESULTS: When CD45(-)TER119(-) side-scatter(low) GFP(high) cells were sorted, alpha-fetoprotein-positive immature endoderm-characterized cells, having high growth potential, were present in this population. Clonal analysis and electron microscopic evaluation revealed that each single cell of this population could differentiate not only into hepatocytes, but also into biliary epithelial cells, showing their bilineage differentiation activity. When surface markers were analyzed, they were positive for Integrin-alpha6 and -beta1, but negative for c-Kit and Thy1.1. CONCLUSIONS: Combination of GFP-transgenic mice and fluorescence-activated cell sorting enabled purification of hepatic progenitor cells from adult mouse liver. Further analysis of this population may lead to purification of their human correspondence that would be an ideal cell-source candidate for regenerative medicine.

Enrichment of hepatic progenitor cells from adult mouse liver.

Hepatic progenitor cells (HPCs) have been characterized in several drug-treated rodent models and in the fetal liver; however, their properties have not been fully clarified in the normal adult liver, presumably because of their relatively small population and the existence of mature hepatocytes. In an attempt to resolve this issue, we developed a new enrichment system for HPCs using their cell aggregate formation properties. Nonparenchymal cells (NPCs) derived from enzymatically digested liver cells in normal adult mouse liver were treated in a hypoxic 2-hour suspension culture under constant shaking. This procedure resulted in cell aggregate formation and almost complete elimination of mature hepatocytes. Cell aggregates were formed only in Ca(2+)-containing medium, suggesting cadherin-dependent cell-cell adhesion. In these cell aggregates, 95% consisted of vascular endothelial cells that expressed VE-cadherin. The remaining 5% consisted of rapidly proliferating, small epithelial cells that expressed alpha-fetoprotein (AFP), E-cadherin, and albumin but not cytokeratin 19 (CK19), alpha-smooth muscle actin, or VE-cadherin. These results are consistent with an immature hepatic cell phenotype. When these immature hepatic cells were cultured with 10(-7) mol/L dexamethasone and 1% dimethyl sulfoxide, the de novo expression of mature hepatocyte markers such as tryptophan-2,3-dioxygenase (TO) was induced concomitantly with the induction of morphologic characteristics such as mitochondria- and peroxisome-rich cytoplasm and bile canaliculi formation. In conclusion, our methodology allows the enrichment of immature hepatic cells from the normal adult mouse. These cells are capable of growth and maturation along the hepatocyte lineage, indicating that these cells are HPCs.

Thy1-positive mesenchymal cells promote the maturation of CD49f-positive hepatic progenitor cells in the mouse fetal liver.

Previously, we reported a system to enrich mouse fetal hepatic progenitor cells (HPCs) by forming cell aggregates. In this study, we sorted two cell populations, CD49f(+)Thy1(-)CD45(-) cells (CD49f-positive cells) and CD49f(+/-)Thy1(+)CD45(-) cells (Thy1-positive cells), from the cell aggregates using a flow cytometer. CD49f-positive cells stained positive for endodermal specific markers such as alpha-fetoprotein (AFP), albumin (ALB), and cytokeratin 19 (CK19), and are thus thought to be HPCs. However, Thy1-positive cells were a morphologically heterogeneous population; reverse-transcription polymerase chain reaction (RT-PCR) and immunocytochemical analyses revealed the expression of mesenchymal cell markers such as alpha-smooth muscle actin, desmin, and vimentin, but not of AFP, ALB, or CK19. Therefore, Thy1-positive cells were thought to be of a mesenchymal lineage. When these two cell populations were co-cultured, the CD49f-positive colonies matured morphologically and stored a significant amount of glycogen. Furthermore, real-time RT-PCR demonstrated an increased expression of tyrosine amino transferase and tryptophan oxygenase mRNA, and transmission electron microscopy confirmed that co-cultured cells produced mature hepatocytes. However, when CD49f-positive cells were cultured alone or when the two populations were cultured separately, the CD49f-positive cells did not mature. These results indicate that CD49f-positive cells are primitive hepatic endodermal cells with the capacity to differentiate into hepatocytes, and that Thy1-positive cells promote the maturation of CD49f-positive cells by direct cell-to-cell contact. In conclusion, we were able to isolate CD49f-positive primitive hepatic endodermal cells and Thy1-positive mesenchymal cells and to demonstrate the requirement of cell-to-cell contact between these cell types for the maturation of the hepatic precursors.

Commitment of bone marrow cells to hepatic stellate cells in mouse.

BACKGROUND/AIMS: Recently, several cells found within the liver have been reported to derive from bone marrow (BM). This study sought to examine the commitment of BM cells to hepatic stellate cell (HSC) lineage in mouse liver. METHODS: We transplanted BM cells from green fluorescent protein (GFP) transgenic mice into age-matched C57BL/J mice. Hepatic nonparenchymal cells were isolated from the livers of BM-transplanted mice using density gradient centrifugation with Nycodenz. The expression of lineage markers by the isolated cells was evaluated by RT-PCR and immunostaining. We then examined the histology of liver tissues obtained from BM-transplanted mice with and without carbon tetrachloride-induced injury. RESULTS: GFP-expressing cells with intracytoplasmic lipid droplets comprised 33.4 +/- 2.3% of the cells isolated by density gradient centrifugation. These cells expressed the HSC lineage markers, such as desmin and glial fibrillary acidic protein (GFAP), by both RT-PCR and immunostaining. During a 7-day culture, GFP-positive cells began to express alpha-smooth muscle actin, a marker of activated HSC. In the liver of BM-transplanted mice, GFP-positive nonparenchymal cells expressed GFAP and extended their process around hepatocytes. Upon liver injury, these cells also co-expressed desmin and alpha-smooth muscle actin. CONCLUSIONS: Nonparenchymal cells, derived from transplanted BM, acquired HSC characteristics in both quiescent and activated states.

Contribution of bone marrow cells to liver regeneration after partial hepatectomy in mice.

BACKGROUND/AIMS: We examined whether bone marrow (BM) cells can commit to liver-consisting cells during liver regeneration after partial hepatectomy, using mice transplanted with green fluorescent protein (GFP) positive BM from GFP transgenic mice. METHODS: Partial hepatectomy or sham operation was performed. Lineage marker analysis of GFP positive liver cells was by immunostaining and flow cytometry. DiI-labeled acetylated low-density lipoprotein uptake or microsphere phagocytosis was examined in vitro. Lineage marker expression in BM and peripheral blood (PB) cells, and the vascular endothelial growth factor (VEGF) concentration in the liver were also examined. RESULTS: In hepatectomized mice, significantly more GFP positive cells participated in liver sinusoid than in sham-operated mice, expressing CD31 but not albumin. The percentage of cells that incorporated acetylated low-density lipoprotein but not microspheres was 69.5+/-3.4%, while 28.3+/-2.6% incorporated both, revealing sinusoidal endothelial and Kupffer cells, respectively. Increased expression of the CD31 and CD16/CD32 on GFP positive liver cells was also detected. The elevation of the VEGF concentration during liver regeneration and the increase in the CD34 and Flk-1 expression in the liver, BM, and PB cells suggested endothelial progenitor cell mobilization. CONCLUSIONS: GFP cell-marking provided direct evidence of the BM cells participation in liver regeneration after hepatectomy, where the majority was committed to sinusoidal endothelial cells probably through endothelial progenitor cell mobilization.

Establishment of a highly efficient gene transfer system for mouse fetal hepatic progenitor cells.

Because of a donor shortage problem in liver transplantation, cell transplantation has been anticipated as a useful bridge or substitute therapy, and has necessitated the development of cell sources other than donated organs. Therefore, the use of fetal hepatic progenitor cells (HPCs) is now being focused on. In this study, we intended to establish an efficient ex vivo nonviral gene-transfer system using a newly developed isolation and culture system for mouse fetal HPCs. Fetal HPCs, characterized using immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) for lineage markers, were collected from E13.5 Balb/c mice using change in size because of cell aggregation by their homophilic cell-to-cell binding occurring during suspension culture. Optimal conditions for culture and ex vivo gene transfection for fetal HPCs were determined by (3)H-thymidine incorporation and the expression efficacy of transfected red fluorescent protein (DsRed) gene in different culture media. The optimum timing for gene transfection was also evaluated. To evaluate the in vivo expression of the transferred gene, DsRed-transferred fetal HPCs were transplanted into 70% partially hepatectomized allogenic mice. The highest efficacy of DsRed gene transfection into fetal HPCs in vitro (45% +/- 12.3%) was achieved with culture media, which also enabled the highest (3)H-thymidine incorporation, containing the deleted form of hepatocyte growth factor (dHGF) and insulin, and when transfection was performed immediately after isolation. In vivo DsRed expression in fetal HPCs was maintained concomitantly with albumin expression even after HPC transplantation. In conclusion, we established a highly efficient in vitro gene transfer system for mouse fetal HPCs using a newly developed isolation and culture system.