Mutation Spectrum of EGFR From 21,324 Chinese Patients With Non-Small Cell Lung Cancer (NSCLC) Successfully Tested by Multiple Methods in a CAP-Accredited Laboratory.

Genotyping epidermal growth factor receptor (EGFR) gene in patients with advanced non-small cell lung cancers (NSCLC) is essential for identifying those patients who may benefit from targeted therapies. Systemically evaluating EGFR mutation detection rates of different methods currently used in clinical setting will provide valuable information to clinicians and laboratory scientists who take care of NSCLC patients. This study retrospectively reviewed the EGFR data obtained in our laboratory in last 10 years. A total of 21,324 NSCLC cases successfully underwent EGFR genotyping for clinical therapeutic purpose, including 5,244 cases tested by Sanger sequencing, 13,329 cases tested by real-time PCR, and 2,751 tested by next-generation sequencing (NGS). The average EGFR mutation rate was 45.1%, with 40.3% identified by Sanger sequencing, 46.5% by real-time PCR and 47.5% by NGS. Of these cases with EGFR mutations identified, 93.3% of them harbored a single EGFR mutation (92.1% with 19del or L858R, and 7.9% with uncommon mutations) and 6.7% harbored complex EGFR mutations. Of the 72 distinct EGFR variants identified in this study, 15 of them (single or complex EGFR mutations) were newly identified in NSCLC. For these cases with EGFR mutations tested by NGS, 65.3% of them also carried tumor-related variants in some non-EGFR genes and about one third of them were considered candidates of targeted drugs. NGS method showed advantages over Sanger sequencing and real-time PCR not only by providing the highest mutation detection rate of EGFR but also by identifying actionable non-EGFR mutations with targeted drugs in clinical setting.

Is there a primitive reflex residue underlying Marcus Gunn Syndrome? Rat electrophysiology.

AIM: To make an electrophysiological demonstration of a possible jaw muscle afferents-oculomotor neural pathway that was proposed by our previous works on rats, which substantiates an early "release hypothesis" on pathogenesis of human Marcus Gunn Syndrome (MGS). METHODS: Extracellular unit discharge recording was applied and both orthodromic and spontaneous unitary firing were recorded in the oculomotor nucleus (III), and the complex of pre-oculomotor interstitial nucleus of Cajal and Darkschewitsch nucleus (INC/DN), following electric stimulation of the ipsilateral masseter nerve (MN) in rats. RESULTS: Extracellular orthodromic unit discharges, with latencies of 3.7±1.3 and 4.7±2.9ms, were recorded unilaterally in the III, and the INC/DN neurons, respectively. Spontaneous unit discharges were also recorded mostly in the INC/DN and less frequently in the III. Train stimulation could prompt either facilitation or inhibition on those spontaneous unit discharges. The inhibition pattern of train stimulation on the spontaneous discharging was rather different in the III and INC/DN. A slow inhibitory pattern in which spontaneous firing rate decreased further and further following repeated train stimulation was observed in the III. While, some high spontaneous firing rate units, responding promptly to the train stimuli with a short-term inhibition and recovered quickly when stimuli are off, were recorded in the INC/DN. However, orthodromic unit discharge was not recorded in the III and INC/DN in a considerable number of experiment animals. CONCLUSION: A residual neuronal circuit might exist in mammals for the primitive jaw-eyelid reflex observed in amphibians, which might not be well-developed in all experimental mammals in current study. Nonetheless, this pathway can be still considered as a neuroanatomic substrate for development of MGS in some cases among all MGS with different kind of etiology.

Is Marcus Gunn jaw winking a primitive reflex? Rat neuroanatomy.

AIM: To investigate a possible trigeminal proprioceptive-oculomotor neural pathway and explore possible synaptic connections between neurons in this pathway. Attempt to bring a new insight to mechanism of Marcus Gunn syndrome (MGS). METHODS: Anterograde and retrograde tract tracing was applied and combined with immunofluorescent stain in rats. After electrophysiological identifying mesencephalic trigeminal nucleus (Vme) neurons, intracellular injection of tracer was performed to trace axon trajectory. RESULTS: Following injections of anterograde tracers into the Vme, labeled terminals were observed ipsilateral in oculomotor and trochlear nuclei (III/IV), as well as in their premotor neurons in interstitial nucleus of Cajal and Darkschewitsch nucleus (INC/DN). Combining with choline acetyltransferase (ChAT) immunofluorescent stain, it showed that Vme projecting terminals contact upon ChAT positive III/IV motoneurons under confocal microscope. By retrograde labeling premotor neurons of the III, it showed that Vme neuronal terminals contact with retrogradely labeled pre-oculomotor neurons in the INC/DN. Axons of intracellularly labeled Vme neurons that respond to electric stimuli of the masseter nerve traveled into the ipsilateral III. CONCLUSION: There may exist a trigeminal proprioceptive-oculomotor system neural circuit in the rat, which is probably related to vertical-torsional eye movements. Possible association of this pathway with MGS etiology was discussed.

Electromyography and Fos immunostaining study establish a possible functional link between trigeminal proprioception and the oculomotor system in rats.

The objective of this study was to explore whether there was a functional link between trigeminal proprioception and the oculomotor system mediated through jaw muscle afferents. Electromyography (EMG) was undertaken of the levator palpebrae (LP) and superior rectus (SR), and Fos expression was detected in the brainstem following consecutive down-stretching of the lower jaw at 2-4 Hz in rats. Retrograde tracing was undertaken of the interstitial nucleus of Cajal and Darkschewitsch nucleus (INC/DN) pre-oculomotor neurons. EMG-like responses were recorded from the LP/SR during down-stretching of the lower jaw at 2-4 Hz in 3 out of 11 rats. Fos expression was induced by consecutive down-stretching of the lower jaw at 2-4 Hz for 20-30 seconds. Interestingly, Fos expression was distributed mainly in the bilateral INC/DN area. We also examined Fos-like immunoreactivity in central mesencephalic and paramedian pontine reticular formation that harbors premotor neurons controlling horizontal eye movement, but no Fos-like staining was observed therein. By injection of retrograde tracers into the oculomotor nucleus combined with Fos immunostaining, double labeled pre-oculomotor neurons were visualized to distribute in the INC/DN. In conclusions, there may exist a trigeminal proprioceptive - oculomotor system neural circuit through jaw muscle afferents in rats. Judging from Fos distribution pattern, this pathway might be related to vertical and torsional eye movements.

Jaw muscle spindle afferents coordinate multiple orofacial motoneurons via common premotor neurons in rats: an electrophysiological and anatomical study.

Jaw muscle spindle afferents (JMSA) in the mesencephalic trigeminal nucleus (Vme) project to the parvocellular reticular nucleus (PCRt) and dorsomedial spinal trigeminal nucleus (dm-Vsp). A number of premotor neurons that project to the trigeminal motor nucleus (Vmo), facial nucleus (VII) and hypoglossal nucleus (XII) are also located in the PCRt and dm-Vsp. In this study, we examined whether these premotor neurons serve as common relay pool for relaying JMSA to multiple orofacial motoneurons. JMSA inputs to the PCRt and dm-Vsp neurons were verified by recording extracellular responses to electrical stimulation of the caudal Vme or masseter nerve, mechanical stimulation of jaw muscles and jaw opening. After recording, biocytin in recording electrode was inotophorized into recording sites. Biocytin-Iabeled fibers traveled to the Vmo, VII, XII, and the nucleus ambiguus (Amb). Labeled boutons were seen in close apposition with Nissl-stained motoneurons in the Vmo, VII, XII and Amb. In addition, an anterograde tracer (biotinylated dextran amine) was iontophorized into the caudal Vme, and a retrograde tracer (Cholera toxin B subunit) was delivered into either the VII or Xll to identify VII and XII premotor neurons that receive JMSA input. Contacts between labeled Vme neuronal boutons and premotor neurons were observed in the PCRt and adjacent dm-Vsp. Confocal microscopic observations confirmed close contacts between Vme boutons and VII and XII premotor neurons. This study provides evidence that JMSA may coordinate activities of multiple orofacial motor nuclei, including Vmo, VII, XII and Amb in the brainstem via a common premotor neuron pool.

Nodular histiocytic aggregates in the greater omentum of patients with ovarian cancer.

Nodular histiocytic aggregate (NHA) of the omentum is a rare benign proliferative process composed predominantly of histiocytes with scattered mesothelial cells. NHA is a differential diagnosis for neoplasms or metastatic tumors in cancer patients. To further clarify this clinical pitfall issue, the authors investigated surgical samples of the greater omentum from 96 patients with gastrointestinal malignancies and 53 patients with gynecologic neoplasms. Visible NHA of greater omentum was identified in 3 patients with ovarian neoplasms (borderline mucinous cystadenoma, low-grade papillary serous cystadenocarcinoma, and juvenile granulosa-cell tumor) but in none of the patients with gastrointestinal malignancies. Similar lesion was also identified on the cell blocks from peritoneal washings in 1 of the 3 patients. Grossly, the lesions formed small yellow-red nodules on the greater omentum, and the NHA lesion was also found diffusely on the surface of the appendix and fallopian tubes in 2 of the 3 patients. Histological study showed that typical NHA changes over an inflammatory background, which may indicate that NHA is a consequence of a chronic inflammatory process of omentum. The predominant infiltration of T lymphocytes in the NHA lesions indicates that the aggregation of histiocytes may be related to the activation of T-cell immunity. This report has first demonstrated visible NHA in the greater omentum of patients with ovarian malignancies, and awareness of this entity should be brought to clinicians to avoid misdiagnosis.

Action potential-triggered somatic exocytosis in mesencephalic trigeminal nucleus neurons in rat brain slices.

The neurons in the mesencephalic trigeminal nucleus (MeV) play essential roles in proprioceptive sensation of the face and oral cavity. The somata of MeV neurons are generally assumed to carry out neuronal functions but not to play a direct role in synaptic transmission. Using whole-cell recording and membrane capacitance (C(m)) measurements, we found that the somata of MeV neurons underwent robust exocytosis (C(m) jumps) upon depolarization and with the normal firing of action potentials in brain slices. Both removing [Ca(2+)](o) and buffering [Ca(2+)](i) with BAPTA blocked this exocytosis, indicating that it was completely Ca(2+) dependent. In addition, an electron microscopic study showed synaptic-like vesicles approximated to the plasma membrane in somata. There was a single Ca(2+)-dependent releasable vesicle pool with a peak release rate of 1912 fF s(-1). Importantly, following depolarization-induced somatic exocytosis, GABA-mediated postsynaptic currents were transiently reduced by 31%, suggesting that the somatic vesicular release had a retrograde effect on afferent GABAergic transmission. These results provide strong evidence that the somata of MeV neurons undergo robust somatic secretion and may play a crucial role in bidirectional communication between somata and their synaptic inputs in the central nervous system.

Unraveling a masticatory - oculomotor neural pathway in rat: Implications for a pathophysiological neural circuit in human?

Anterograde tracers were injected into the mesencephalic trigeminal nucleus (Vme) in pons, labeled axons and terminals were observed in ipsilateral oculomotor (III) and trochlear (IV) nuclei, as well as in interstitial nucleus of Cajar and Darkschewitsch nucleus (INC/DN), the well-known premotor nuclei to the III/IV, but not in abducens nucleus and central mesencephalic and paramedian pontine reticular formation (CMRF/PPRF). Retrogradely labeled INC/DN neurons do ensue from injection of tracers into the III. Confocal microscopy revealed labeled Vme axonal terminals contact with labeled pre-oculomotor neurons in the INC/DN. In response to electrical stimulation of trigeminal nerve root (TR) jaw muscle branches, which contains peripheral processes of the jaw muscle spindle, extracellular unit discharges were recorded in the ipsilateral III/IV and INC/DN. Electromyography (EMG) was also recorded from superior rectus (SR) and levator palpebrae (LP) following electrical stimulation of the TR. Moreover, stimulation of the TR induced Fos expression in the INC/DN pre-oculomotor neurons, but not in CMRF/PPRF that harbors horizontal eye moving premotor neurons. By injection of retrograde tracers into the III combined with Fos immunostain, double labeled pre-oculomotor neurons were observed in the INC/DN. About 80% of retrogradely labeled III premotor neurons express Fos. These results suggest a neural pathway from the masticatory Vme neurons to the oculomotor system that is probably involved exclusively in vertical and torsional eye movement as well as eyelid retraction. The potential relationship between this pathway and Marcus Gunn Syndrome (MGS), a congenital jaw-winking syndrome, was discussed.

Successful management of hemopericardium and cardiac tamponade secondary to occult malignancy and anticoagulation.

In patients presenting with pericarditis or pericardial effusion without known malignancy, the likelihood of finding previously undiagnosed cancer in different publications typically ranges from 4% to 7%. Cardiac tamponade due to malignant pericardial effusion is thus a rare clinical entity and often acutely life threatening. The present report describes an unusual case of large pericardial bleeding causing tamponade in the setting of fondaparinux anticoagulation, heterozygous factor V Leiden mutation and eventual discovery of meta-static adenocarcinoma.

Serous papillary cystadenocarcinoma arising from autografted ovary of the abdominal wall.

A 58-year-old-woman developed a serous papillary cystadenocarcinoma between the fascia and peritoneum of the left abdominal wall. The patient had undergone bilateral oophorectomy for serous cystadenoma 17 years earlier and her residual normal ovarian parenchyma had also been transplanted to the abdominal wall. Grossly and microscopically, the current tumor arises from the autografted ovarian parenchyma. Literature review indicates that carcinoma arising from autografted ovarian tissue is extremely rare.

Pathology and FDG PET correlation of residual lymph nodes in head and neck cancer after radiation treatment.

BACKGROUND: This study determines if postradiotherapy [18F]fluorodeoxyglucose positron emission tomography (FDG PET) can predict the pathology status of residual cervical lymph nodes in patients undergoing definitive radiotherapy for head and neck squamous cell carcinoma (HNSCC). METHODS: Patients with stage N2 or higher HNSCC underwent PET and CT imaging after definitive radiotherapy. Patients with radiographically persistent lymphadenopathy underwent either neck dissection or fine needle aspiration (FNA) of the lymph nodes under ultrasound guidance. PET scan results were correlated with the pathologic findings of the residual lymphadenopathy. RESULTS: Twenty-four hemi-necks in 23 patients with residual lymphadenopathy had neck dissection or FNA. The pathology correlated strongly with the post-RT FDG PET studies. All patients with a negative post-RT FDG PET and those with a maximum standardized uptake value (SUVmax) of less than 3.0 in the post-RT FDG PET were found to be free from residual viable tumor. Using a SUVmax of less than 3.0 as the criterion for a negative FDG PET study, the sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 84.2%, 62.5%, and 100%, respectively. CONCLUSIONS: A negative post-RT FDG PET is very predictive of negative pathology in the residual lymph node after definitive radiotherapy for advanced HNSCC. A prospective clinical trial is warranted to determine if neck dissection can be withheld in these patients.

Neuronal circuitry and synaptic organization of trigeminal proprioceptive afferents mediating tongue movement and jaw-tongue coordination via hypoglossal premotor neurons.

The neural framework and synaptic organization of trigeminal proprioceptive afferent-mediated jaw-tongue coordination were studied in rats using multiple electrophysiological and neuroanatomical approaches. Electrostimulation of the masseter nerve evoked short-latency responses (5.86 +/- 2.59 ms) in hypoglossal premotor pools including the parvocellular (PCRt) and intermediate (IRt) reticular nuclei and the dorsomedial part of the spinal trigeminal nucleus oralis (Vodm) and interpolaris (Vidm). Biocytin-labelled axon terminals from these areas traveled into the hypoglossal nucleus (XII) and contacted motoneurons. Double labelling of biotinylated dextran amine (BDA) tracing and cholera toxin B (CTB) transport demonstrated that labelled axons and terminals from the mesencephalic trigeminal nucleus (Vme) overlapped with XII premotor neurons in the alpha division and in PCRt, IRt, Vodm and Vidm. Confocal microscopic observations revealed that Vme terminals closely contacted XII premotor neurons. Dual labelling of intracellular neurobiotin staining of jaw-muscle spindle afferents (JMSAs) combined with horseradish peroxidase (HRP) retrograde transport revealed that 498 JMSA boutons apposed to 146 HRP-labelled premotor neurons. Electron microscopic observations demonstrated that 127 JMSA boutons made both axodendritic (68%) and axosomatic (32%) synapses with XII premotor neurons. Eighty-three per cent of synapses were asymmetric and the rest (17%) were symmetric. Thirty-nine per cent of JMSA boutons received presynaptic contacts from P-type terminals. Varieties of synaptic organizations were found. These results provide evidence that trigeminal proprioceptive afferents mediate jaw-tongue coordination through XII premotor neurons. Ultrastructural findings demonstrated that synapses between JMSA boutons and XII premotor neurons are predominantly excitatory, and synaptic transmission to XII motoneurons is modified on XII premotor neurons by presynaptic mechanisms. These frameworks and synaptic organizations are most probably the neural substrate for trigeminal proprioceptive afferent-mediated jaw-tongue coordination.

Synaptic organization of monosynaptic connections from mesencephalic trigeminal nucleus neurons to hypoglossal motoneurons in the rat.

Synaptological characteristics of synapses between axonal boutons of the trigeminal mesencephalic nucleus (Vme) neurons and the hypoglossal nucleus (XII) motoneurons (MNs) were studied using biotinylated dextran amine (BDA) anterograde labeling combined with horseradish peroxidase (HRP) retrograde transport in the rat. BDA was initially iontophoresed into Vme unilaterally and 7 days later HRP was injected into the anterior two-thirds of the ipsilateral tongue. After histochemical reactions, BDA anterogradely labeled boutons were seen to appose closely to somata and dendrites of HRP retrogradely labeled MNs in XII by light microscopy. A total of 212 BDA-labeled Vme boutons were examined ultrastructurally, which had an average diameter of 1.3 +/- 0.4 microm and contain small clear spherical vesicles. Eighty-eight percent of Vme boutons (187/212) synapsed on dendrites of HRP-labeled XII MNs. Twenty-five Vme boutons (25/212, 12%) made synapses with somata of HRP-labeled XII MNs. Thirty-five percent (74/212) of BDA-labeled Vme boutons were also contacted by unlabeled P-type terminals. Presynaptic P-type terminals contained spherical (47%, 35/74), pleomorphic (43%, 32/74), and flattened (10%, 7/74) synaptic vesicles. Thus, P-type terminals (as a presynaptic element), BDA-labeled Vme boutons, and XII MNs constitute axoaxodendritic and axoaxosomatic synaptic triads. There are four types of synaptic microcircuits in XII neuropil: synaptic convergence, synaptic divergence, presynaptic inhibition synaptic circuits, and feedforward regulation circuits. This detailed ultrastructure examination of the synaptic organization between Vme neurons and XII MNs provides insights into the synaptic mechanisms of the trigeminal proprioceptive afferents involved in the jaw-tongue reflex and coordination during oral motor behaviors.

Ultrastructural features of synapse from dorsal parvocellular reticular formation neurons to hypoglossal motoneurons of the rat.

The dorsal parvocellular reticular formation (PCRt) receives projection of the trigeminal mesencephalic nucleus neurons. It contains the dorsal group of interneurons that integrate and coordinate activity of the oral motor nuclei. Ultrastructural features of synaptic connection from the dorsal PCRt neurons to the motoneurons of the hypoglossal nucleus (XII) were examined at both the light and electron microscopic levels in rats. Biotinylated dextran amine (BDA) was initially iontophoresed into the dorsal part of PCRt unilaterally. Seven days later horseradish peroxidase (HRP) was injected into the body of the tongue. After histochemical reaction for visualization of HRP and BDA, the BDA-labeled fibers and terminals were seen distributing bilaterally in XII with ipsilateral predominance. BDA-labeled terminals were closely apposed upon HRP retrogradely labeled somata and dendrites of the XII motoneurons. A total of 1408 BDA-labeled boutons were examined ultrastructurally, which had mean size of 1.22+/-0.37 microm in diameter. Five hundred-ninety three of these boutons in both the ipsilateral (n=401) and contralateral (n=192) XII were seen to synapse on both the dendrites and somata of HRP-labeled motoneurons. The vast majorities of synapses were axodendritic (98%, 580/593), while 2% of them were axosomatic. Of the 1408 BDA-labeled boutons, 69.6% of them were S-type boutons containing small clear and spherical synaptic vesicles and 30.4% of them were PF-type boutons containing pleomorphic and flattened synaptic vesicles. Approximately 64% of synapses between BDA-labeled boutons and HRP-labeled motoneurons were asymmetric, and 33% of synapses were symmetric. No axoaxodendritic or axoaxosomatic synaptic triad was observed. The present study illustrated the anatomical pathway and synaptological characteristics of neuronal connection between the dorsal PCRt premotor neurons and the XII motoneurons. Its functional significance in coordinating activity of XII motoneurons during oral motor behaviors has been discussed.

Orexin B immunoreactive fibers and terminals innervate the sensory and motor neurons of jaw-elevator muscles in the rat.

The relationship between orexinergic terminals and the sensory and motor neurons of jaw-elevator muscles was examined by means of anti-orexin B (OXB) immunohistochemistry combined with horseradish peroxidase (HRP) retrograde tracing in the rat. HRP was initially injected into the jaw-elevator muscles; 48 h later the animals were sacrificed and fixed for HRP reaction and anti-OXB immunohistochemistry. OXB-like terminals were observed to distribute in the trigeminal mesencephalic nucleus (Vme) and the trigeminal motor nucleus (Vmo) where they closely contact the Vme neuronal somata and the Vmo neuronal somata and dendrites retrogradely labeled with HRP. The results of this study provide anatomical evidence of a direct OXB orexinergic innervation of the sensory and motor neurons controlling jaw-elevator muscles involved in mastication. Its functional significance related to the feeding behavior and bruxism is discussed.