Picoliter‐Droplet Digital Polymerase Chain Reaction‐Based Analysis of Cell‐Free Plasma DNA to Assess EGFR Mutations in Lung Adenocarcinoma That Confer Resistance to Tyrosine‐Kinase Inhibitors

Purpose.

The objective of this study was to evaluate the utility of analyzing cell-free plasma DNA (cfDNA) by picoliter-droplet digital polymerase chain reaction (ddPCR) to detect EGFR mutations that confer resistance to tyrosine-kinase inhibitors (TKIs) used for treatment of lung adenocarcinoma (LADC).

Experimental design.

Thirty-five LADC patients who received epidermal growth factor receptor (EGFR)-TKI therapy, including ten who received tumor rebiopsy after development of resistance, were subjected to picoliter-ddPCR-cfDNA analysis to determine the fraction of cfDNA with TKI-sensitive (L858R and inflame exon 19 deletions) and -resistant (i.e., T790M) mutations, as well as their concordance with mutation status in rebiopsied tumor tissues.

Results.

cfDNA samples from 15 (94%) of 16 patients who acquired resistance were positive for TKI-sensitive mutations. Also, 7 (44%) were positive for the T790M mutation, with fractions of T790M (+) cfDNA ranging from 7.4% to 97%. T790M positivity in cfDNA was consistent in eight of ten patients for whom rebiopsied tumor tissues were analyzed, whereas the remaining cases were negative in cfDNA and positive in rebiopsied tumors. Prior to EGFR-TKI therapy, cfDNAs from 9 (38%) and 0 of 24 patients were positive for TKI-sensitive and T790M mutations, respectively. Next-generation sequencing of cfDNA from one patient who exhibited innate resistance to TKI despite a high fraction of TKI-sensitive mutations and the absence of the T790M mutation in his cfDNA revealed the presence of the L747P mutation, a known driver of TKI resistance.

Conclusion.

Picoliter-ddPCR examination of cfDNA, supported by next-generation sequencing analysis, enables noninvasive assessment of EGFR mutations that confer resistance to TKIs.

Implications for Practice:

Noninvasive monitoring of the predominance of tumors harboring the secondary T790M mutation in the activating mutation in EGFR gene is necessary for precise and effective treatment of lung adenocarcinoma. Because cells harboring the T790M mutation are resistant to epidermal growth factor receptor-tyrosine-kinase inhibitors (TKIs), the predominance of tumor cells harboring the T790M mutations influences the choice of whether to use conventional or next-generation TKIs. Digital polymerase chain reaction-based examination of cfDNA is a promising method; however, its feasibility, including its consistency with examination of rebiopsied tumor tissue, has not been fully proven. Here, picoliter-droplet digital polymerase chain reaction technology is presented as a candidate method for testing cfDNA and assessing the predominance of T790M-mutant tumors.     

Lens epithelial cell death after cataract surgery

PURPOSE: To determine whether lens epithelial cells (LECs) undergo apoptosis during healing after cataract surgery to further characterize the healing process of the postoperative lens capsule. SETTING: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. METHODS: Apoptotic cells were detected in human postoperative lens capsules using the terminal deoxynucleotidyl transferase deoxy-UTP nick-end labeling (TUNEL) method. The effect of exogenous transforming growth factor-beta2 (TGF-beta2) on mouse LEC apoptosis was also examined in an organ-culture system. RESULTS: Three of 17 postoperative specimens contained TUNEL-positive cells. In 2 lens capsules obtained earlier than 10 days, many TUNEL-positive cells, presumably apoptotic LECs, were observed beneath the residual anterior capsule. In cell multilayers in capsule opacification extracted later than 10 days, a few TUNEL-positive cells were seen in 1 specimen; most cells remained negative. In mouse lenses organ-cultured with 1.0 ng/mL TGF-beta2 for 48 hours, TUNEL-positive cells were detected beneath the lens capsule. CONCLUSIONS: Lens epithelial cells undergo apoptosis during healing after cataract surgery, especially in the early phase. Transforming growth factor-beta2 may be a factor inducing apoptosis in in vivo LECs.     

Avoidance learning under delayed shock termination in prenatally x-irradiated rats

Groups of MP1 male albino rats were exposed to x-irradiation of 200 rad on Day 17 of gestation. Irradiated and control rats were trained in a shuttlebox with either immediate or .5-sec delayed termination of both the electric shock (US) and the conditioned stimulus (CS) on escape trials. An avoidance response always produced prompt CS termination. Irradiated rats showed a higher degree of avoidance behavior than controls and, under the delay condition, tended to run rapidly in the CS-US interval.     

Neuron–Glia Interaction in the Suprachiasmatic Nucleus: A Double Labeling Light and Electron Microscopic Immunocytochemical Study in the Rat

The morphological interactions between astroglial and neuronal elements were elucidated in the rat suprachiasmatic nucleus (SCN) by light and electron microscopic immunocytochemistry using antibodies against glial fibrillary acidic protein (GFAP), vasoactive intestinal peptide (VIP) and arginine-vasopressin (AVP). Throughout the SCN, particularly in its ventral portion, GFAP-like-immunoreactive (GFAP-LI) astroglial elements were found. These astrocytes displaying GFAP-like immunoreactivity occasionally contained fairly well-developed organelles. Some of these astrocytes were found as satellite cells in close contact with non-immunoreactive neuronal perikarya and processes. Around the neurons, GFAP-LI astroglial processes were also observed to cover some portions of presynaptic and postsynaptic elements. In addition, these astroglial elements were seen between two neuronal somata and pericytes of blood capillaries as glial endfeet. By double labeling immunoelectron microscopy using antibodies against GFAP/VIP and GFAP/AVP, some portions of VIP-like-immunoreactive or AVP-like-immunoreactive neuronal somata and processes were found to be engulfed by GFAP-LI astroglial processes. The possible functional roles of the morphological interactions between astroglial and neuronal elements are discussed.     

A-W glass ceramic as a bone substitute in cemented hip arthroplasty 15 hips followed 2-10 years

We retrospectively reviewed hip arthroplasties in 13 patients (15 hips), in whom we had used apatite-wol-lastonite (A-W) glass ceramic together with auto- or allograft for augmentation of severe bone deficiency. 11 cemented sockets and 4 stem revisions were included and followed for 2-9.6 years. There were no radiolucent lines between A-W glass ceramic and surrounding bone, and remodeling of the bone graft containing A-W glass ceramic was observed.

No migration of cemented sockets was seen except in 1 case, which was revised. In this case, direct bonding between bone and A-W glass ceramic granules was present histologically. In 4 stem revisions, 5 mm subsidence occurred in 1 case. However, the stem became stable and remodeling of the grafted bone occurred. An artificial bone material, such as A-W glass ceramic, can be used under high-load conditions, because of its good mechanical properties.     

Dose-dependent effects of repeated ketamine administration on muscarinic acetylcholine receptors in the mouse forebrain

To study the effects of repeated ketamine administration (0: saline, 12.5, 25, and 50 mg·kg⁻¹ every 3 days for a total of five times, subcutaneously) on the central muscarinic acetylcholine receptors (mAchRs), receptor binding assays of mAchR were carried out in the forebrain of mice, using [³H]quinuclidinyl benzilate ([³H]QNB) as a ligand. We also examined whether repeated ketamine administration could modify the sensitivity to scopolamine (0.5 mg·kg⁻¹) (a muscarinic antagonist). Repeated ketamine administration produced a significant increase in the receptor density values (Bmax) for [³H]QNB (1520±51 fmol·mg protein⁻¹ for the control group, 1650±43 for the 12.5 mg·kg⁻¹ group, 1966±70 for the 25 mg·kg⁻¹ group and 2064±125 for the 50 mg·kg⁻¹ group) (P<0.05, when the 25 mg·kg⁻¹ and 50 mg·kg⁻¹ groups were compared to the control group) without any change in apparent affinity. Repeated ketamine reduced scopolamine-induced hyperlocomotion at 50 mg·kg⁻¹ (P<0.05). We conclude that repeated ketamine administration produces up-regulation of mAchRs, which is probably associated with the altered Ach transmission of the central nervous system.     

RETRACTED ARTICLE: Dibutyryl cyclic AMP increases the contractility of fatigued diaphragm in dogs

The effects of dibutyryl cyclic AMP (DBcAMP) on the contractility of nonfatigued and fatigued diaphragms were studied in 36 anesthetized and mechanically ventilated dogs. The animals were divided into four groups. In group C₁ (n=8), dogs without fatigue received only Ringer's lactate solution. In group D₁ (n=8), dogs without fatigue were given a continuous infusion of DBcAMP 0.2 mg·kg⁻¹·min⁻¹. In groups C₂ and D₂ (n=10 each), diaphragmatic fatigue was induced by intermittent supramaximal bilateral electrophrenic stimulation at a frequency of 20 Hz applied for 30 min. In group D₂, after producing fatigue, DBcAMP 0.2 mg·kg⁻¹·min⁻¹ was administered. In groups C₂, only Ringer's solution was administered during this period. Diaphragmatic contractility was assessed by measuring the transdiaphragmatic pressure (P_di, cmH₂O). No difference in P_di was observed in groups C₁ and D₁. After diaphragmatic fatigue in groups C₂ and D₂, P_di at low-frequency (20-Hz) stimulation decreased significantly compared with the prefatigue values (group C₂; 9.3±1.9vs 12.5±2.4, group D₂; 9.3±2.1vs 12.5±2.6; mean±SD;P<0.05), whereas no change in P_di was observed at high-frequency (100-Hz) stimulation. In group D₂, P_di at both stimuli increased significantly with an infusion of DBcAMP compared with the fatigue values (20 Hz; 13.3±3.3vs 9.3±2.1, 100 Hz; 23.4±3.6vs 21.3±3.2;P<0.05). In group C₂, the speed of recovery from fatigue was relatively slower at 20-Hz stimulation than at 100-Hz stimulation. It is concluded that DBcAMP increases the contractility of fatigued diaphragm, but that this agent does not affect the contractility of nonfatigued diaphragm.