Can EGFR mutation status be reliably determined in pre‐operative needle biopsies from adenocarcinomas of the lung?

The identification of EGFR mutations in non‐small‐cell lung cancer is important for selecting patients, who may benefit from treatment with EGFR tyrosine kinase inhibitors. The analysis is usually performed on cytological aspirates and/or histological needle biopsies, representing a small fraction of the tumour volume. The aim of the present investigation was to evaluate the diagnostic performance of this molecular test. We retrospectively included 201 patients with primary adenocarcinoma of the lung. EGFR mutation status (exon 19 deletions and exon 21 L858R point mutation) was evaluated on both pre‐operative biopsies (131 histological and 70 cytological) and on the surgical specimens, using PCR. Samples with low tumour cell fraction were assigned to laser micro‐dissection (LMD). We found nine (4.5%) patients with EGFR mutation in the lung tumour resections, but failed to identify mutation in one of the corresponding pre‐operative, cytological specimens. Several (18.4%) analyses of the pre‐operative biopsies were inconclusive, especially in case of biopsies undergoing LMD and regarding exon 21 analysis. Discrepancy of mutation status in one patient may reflect intra‐tumoural heterogeneity or technical issues. Moreover, several inconclusive results in the diagnostic biopsies reveal that attention must be paid on the suitability of pre‐operative biopsies for EGFR mutation analysis.     

A reverse-flow composite flap in the rat.

Reverse-flow flaps are currently particularly used for the reconstruction of defects of the distal part of the extremities. Despite their common usage there have been many reports of postoperative complications, especially resulting in partial or total flap necrosis. There is insufficient knowledge of flap haemodynamics, physiology and wound healing properties in reverse-flow flaps. Development of the proper experimental models is needed to investigate these issues. The purpose of this study was to describe a new reverse-flow flap model in the rat. A total of 20 adult Wistar rats weighing 200-250 g were used in this experiment. In five rats, the vascular anatomy of the auricle of the rat was determined by anatomic dissection and microangiography. In the experimental group (N=5), 1x1 cm reverse-flow composite flaps were harvested as a semi-island shape, based on the distal course of the medial branch of the anterior auricular artery. In the control group, consisting of five rats, the flap was designed and raised based on the proximal course of the medial auricular artery, again in a semi-island shape. In the remaining five animals, a square-shaped composite tissue of the whole layer of the auricle, 1x1 cm in size, was harvested dividing all the bases circumferentially. The composite tissue was replaced in situ. While the former was considered a conventional antegrade-flow flap subgroup, the latter was designated as a graft subgroup. All flaps were replaced in situ. The survival of the flap was evaluated on postoperative day 7 by direct observation and microangiography. The skin island of all the reverse-flow flaps and conventional antegrade-flow flaps survived completely giving a success rate of 100%, whereas all grafts in the control group underwent complete necrosis. Microangiographic studies revealed the vascularity of the reverse-flow and antegrade-flow flaps, identifying the course of the auricular arteries. In conclusion, with its evident advantages of easy to design and harvesting, reliable survival pattern and consistent vascular structure, our new flap model will provide a means for future studies on flap haemodynamics, physiology in reverse-flow flaps.     

Production of chick interferon by reactivating chick erythrocytes

When chick erythrocytes were fused with mouse L929 or A9 cells and the heterokaryons induced for interferon production on consecutive days, considerable amounts of mouse interferon were produced every day. Small amounts of chick interferon were produced 2 to 3 days after fusion, coincident with the appearance of a chick enzyme, appearance of nucleoli and increase in chick cell nuclear diameter.     

A key requirement for synaptic Reelin signaling in ketamine-mediated behavioral and synaptic action

Ketamine is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist that produces rapid antidepressant action in some patients with treatment-resistant depression. However, recent data suggest that ∼50% of patients with treatment-resistant depression do not respond to ketamine. The factors that contribute to the nonresponsiveness to ketamine’s antidepressant action remain unclear. Recent studies have reported a role for secreted glycoprotein Reelin in regulating pre- and postsynaptic function, which suggests that Reelin may be involved in ketamine’s antidepressant action, although the premise has not been tested. Here, we investigated whether the disruption of Reelin-mediated synaptic signaling alters ketamine-triggered synaptic plasticity and behavioral effects. To this end, we used mouse models with genetic deletion of Reelin or apolipoprotein E receptor 2 (Apoer2), as well as pharmacological inhibition of their downstream effectors, Src family kinases (SFKs) or phosphoinositide 3-kinase. We found that disruption of Reelin, Apoer2, or SFKs blocks ketamine-driven behavioral changes and synaptic plasticity in the hippocampal CA1 region. Although ketamine administration did not affect tyrosine phosphorylation of DAB1, an adaptor protein linked to downstream signaling of Reelin, disruption of Apoer2 or SFKs impaired baseline NMDA receptor–mediated neurotransmission. These results suggest that maintenance of baseline NMDA receptor function by Reelin signaling may be a key permissive factor required for ketamine’s antidepressant effects. Taken together, our results suggest that impairments in Reelin-Apoer2-SFK pathway components may in part underlie nonresponsiveness to ketamine’s antidepressant action.

Major depressive disorder (MDD) is a serious disorder that affects ∼20.6% of the US population and is a leading cause of suicide (1). One crucial problem in treating patients with MDD is an incomplete response rate to medications, where a large fraction of patients do not show a response to primary antidepressant medications (2, 3). Recent clinical findings demonstrate that a subanesthetic dose of ketamine, a noncompetitive N-methyl-d-aspartate receptor (NMDAR) antagonist, produces rapid antidepressant effects within hours in some patients with treatment-resistant depression or MDD (4–6). However, ∼50% of patients with treatment-resistant depression do not respond to ketamine (7), and factors involved in the nonresponsiveness to ketamine remain unclear.The hippocampus is a brain region that has been linked to the pathophysiological changes in MDD. Patients with MDD show a decrease in hippocampal volume and function (8–12). In contrast, MDD patients treated with classic antidepressants have a reversal in hippocampal volume changes along with an improvement in hippocampus-dependent cognitive function (13–15). Previous preclinical studies have shown animal models of depression also exhibit a decrease in hippocampal volume and function (13), and hippocampal synaptic functional enhancement is required to mediate antidepressant responses (16–18). This enhancement of hippocampal function has been suggested to be a key requirement to exert an antidepressant response.Ketamine induces rapid molecular changes that elicit synaptic plasticity in the hippocampus (16, 19–22). Specifically, ketamine rapidly generates synaptic potentiation of field excitatory postsynaptic potentials (fEPSPs) in CA3–CA1 synapses in the hippocampus (ketamine potentiation) by inducing the rapid translation of brain-derived neurotrophic factor (BDNF) and trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) onto the postsynaptic surface (16, 19, 23, 24). Recent studies have shown that if key factors for the antidepressant effects of ketamine, such as BDNF (16, 25, 26) or AMPA receptors (16, 27), are deleted or blocked, the synaptic potentiation in the hippocampus concurrently disappears, suggesting that the synaptic potentiation underlies ketamine’s antidepressant effects (16, 19).Ketamine-mediated potentiation of fEPSPs in CA3–CA1 synapses has been shown to require a block of NMDAR activation by spontaneous glutamate release. Ketamine produces synaptic potentiation in the presence of tetrodotoxin, which blocks sodium channels, and thereby the generation of action potentials, suggesting that blocking NMDARs activated by the spontaneous presynaptic release is key to producing the synaptic potentiation (19, 21, 28, 29). In agreement with this premise, deletion of Vps10p-tail-interactor-1a (Vti1a) and vesicle-associated membrane protein 7 (VAMP7), which are soluble N-ethylmaleimide–sensitive factor attachment protein receptor proteins selectively involved in spontaneous neurotransmitter release (30, 31) in the CA3 hippocampal region, occluded the ketamine potentiation (32). Collectively, these lines of evidence suggest spontaneous glutamate release, and NMDARs are important factors for ketamine potentiation. Thus, it is possible that if these pre- or postsynaptic components are impaired, ketamine may not produce the synaptic potentiation and antidepressant effects.Reelin is a secreted glycoprotein and acts as a neuromodulator in the adult brain by regulating pre- and postsynaptic machinery. Reelin binds to its receptors, apolipoprotein E receptor 2 (Apoer2) and very-low-density lipoprotein receptor (VLDLR) and increases tyrosine phosphorylation in Disabled-1 (DAB1) (33–35). The Reelin pathway regulates pre- or postsynaptic function through its downstream signaling pathways in the adult brain. In presynaptic terminals, the Reelin-Apoer2 pathway activates phosphoinositide 3-kinase (PI3K) and increases Ca²⁺ release from intracellular stores, which in turn mobilizes VAMP7-containing synaptic vesicles and augments spontaneous release (31). At the postsynaptic sites, Reelin’s binding to Apoer2 reciprocally activates DAB1 and Src family kinases (SFKs). Subsequently, the activated SFKs increase tyrosine phosphorylation in NMDAR subunits, GluN2A and GluN2B (34–37), and increase NMDAR open probability (37–39). Since pre- and postsynaptic components regulated by Reelin have been suggested to be important for ketamine potentiation (16, 19–21, 32), it is conceivable that disrupted Reelin signaling may abrogate the antidepressant action and synaptic plasticity of ketamine.To examine this premise, we used genetically modified mice with a deletion of either Reelin or Apoer2 and investigated changes in antidepressant-like behaviors and synaptic potentiation in the CA1 hippocampal region following ketamine treatment. We also used pharmacological inhibitors to examine the effects of signaling molecules downstream of Reelin-Apoer2, specifically SFKs and PI3K, on ketamine-induced behavioral changes and synaptic plasticity. Lastly, we investigated whether the disruption of ketamine’s effects is due to a requirement for the activation of Reelin-dependent signaling or the impairment of NMDAR function by the disruption of Reelin-dependent signaling. Our results suggest that disruption of the Reelin-Apoer2-SFKs pathway depresses NMDAR function and diminishes ketamine’s use-dependent NMDAR antagonism, thereby rendering synapses nonresponsive to ketamine’s action as well as subsequent antidepressant responses. Taken together, these results provide insight into understanding the cellular and molecular mechanisms underlying ketamine’s antidepressant effects.