The reproductive performance of a sheep flock is dependent on a multitude of complex interacting factors. Attaining optimal flock performance requires information about how the reproductive steps are linked and relate to readily available measurements of the state of the flock. The goal was to use data from nine commercial flocks  to investigate and model the key reproductive steps affecting flock reproductive performance. We also developed a maximum-likelihood based methodology to predict flock ovulation rate based on measurements of the number of fetuses at mid-pregnancy (detected by ultrasound-scanning). The model was used to determine how changes in premating liveweight, age, predicted ovulation rate, number of fetuses at mid-pregnancy, lamb survival and lamb growth rate affect the total lamb liveweight at weaning per ewe exposed to the ram in each flock. The data from the commercial flocks were also used to investigate the role of ewe age and premating liveweight on each reproductive step. Sensitivity analyses were conducted to identify the key reproductive steps affecting flock reproductive performance, with a focus on understanding how these steps vary between flocks. The elasticity for embryo survival was 60% of that for lamb survival for these flocks and the elasticities for ovulation rate were highly variable between flocks (0.16 to 0.50 for mature ewes). This indicates that ovulation rate was near-optimal for some flocks, whereas there was potential to significantly improve flock performance in suboptimal flocks. The elasticity for ewe premating liveweight was highly variable between flocks (−0.03 to 0.84 for mature ewes and −0.18 to 1.39 for ewe lambs), indicating that premating liveweight ranged from optimal to suboptimal between flocks. For these suboptimal farms, the opportunity exists to increase flock performance through improved management of ewe premating liveweight. Reproductive loss was significantly greater in ewe lambs than mature ewes, although the difference is dependent on the stage of reproduction and flock. Predicted ovulation rate was 25% lower for ewe lambs and there was a 30% relative decrease in the predicted embryo survival probability from ovulation to scanning for ewe lambs. There was a 10% relative decrease in lamb survival probability from birth to weaning for ewe lambs and lamb growth rate was 25% lower for ewe lambs. A key driver of sheep farm production is the reproductive performance of the flock . The repWe have previously calculated flock elasticities that describe the relative importance of the effect of average premating ewe liveweight (0.81), average ovulation rate (0.33), variance in ovulation rate (−0.095), embryo survival (0.72), lamb survival (1.03), conception failure (0.35), and average ewe age (0.056) on the total kilograms of lamb liveweight at weaning per ewe exposed to the ram in a research flock that had undergone selection for fecundity . This inThus, the first objective of this study was to investigate how ewe age and premating liveweight affect ovulation rate, number of fetuses at mid-pregnancy, lambing day, lamb survival to weaning and weaning weight in nine New Zealand commercial flocks. The second objective was to assess the role of different parameters on the total weight of lambs weaned per ewe exposed to the ram for this flock. The third objective was to investigate the differences in reproductive performance between mature ewes and ewe lambs. Our primary hypothesis is that there are large between flock differences in reproductive performance and that targets to improve reproductive performance are flock specific.The data consisted of more than 300,000 records from nine New Zealand industry flocks collected from 1986 to 2017. Records were extracted with permission from each of the nine flock owners from the New Zealand Sheep Improvement Limited national performance recording database. Each flock size was typically 1,000 ewes. Liveweight and body condition score (BCS) were measured premating, although liveweight measurements were more abundant than BCS measurements. Furthermore, BCS was measured at ultrasound scanning and weaning (liveweight was not measured at scanning or weaning). Ewes were exposed to a ram as a ewe lamb in all flocks, although body condition score measurements were not obtained for ewe lambs.All ewes were mated under commercial conditions and the rams remained with the ewes for approximately two reproductive cycles (34 d). Ewes were ultrasound-scanned during mid-pregnancy to determine the number of fetuses present. The number of lambs born was not accurately recorded in all years and all flocks. Losses from scanning to lambing are likely 1% to 2% , and theIn general, ewes were culled for age after five lambings, although poor performing  ewes were culled at any age to maintain a high-performing flock with a relatively constant size. Animals were managed and observed under standard commercial conditions and all animals had unrestricted access to pasture and water to meet their metabolic requirements.A) terms are omitted from each equation for the ewe lamb models. Ewe lamb premating liveweight was based on liveweight at 8 mo of age, which is the age that ewe lambs were joined with fertile rams.The model components are outlined in the following sections, which are based on the relationships used to investigate the reproductive performance of a research flock . The repi (for ages 2 to 9) is described by a normal distribution with mean  and standard deviation . The notation i ) even though liveweight is known to also affect embryo survival  on the probability of not getting pregnant to the first cycle isThe model for the effect of ovulation rate (q) denotes the probability of failure for an individual ova]. It was assumed that f = 0.87 and q = 0.92 for each flock  and the standard deviation in gestation length (σg = 3 d). The day of lambing for a ewe that lambs to the first cycle , R is the birth rank (one to four lambs born), W is premating ewe liveweight, S is the lamb sex  with perturbation amplitude where where Birthweight was not measured and the mean birthweight for singles was assumed to be 5.20 kg, for twins was 4.28 kg, for triplets was 3.74 kg, for quadruplets was 3.27 kg and for quintuplets was 3.12 kg . Ram lamModel parameters for lamb survival were estimated with a generalized linear mixed-effects model with a binomial distribution and logit link . Model fP < 0.05 unless stated otherwise. Root mean squared error (RMSE) was used to evaluate the accuracy of models for the distributions in a number of fetuses at scanning and premating liveweight.Models were parameterized for each of the nine farms and all farms combined. Separate models were constructed for mature ewes and ewe lambs. The threshold for significance is v) of 0.1 to 1.5 (31) for age. The perturbations in the different factors were simulated in a causal manner. For example, changes in liveweight included a future cascade of causally related changes in other reproductive variables while, for example, a change in lamb survival would not reflect changes in prior correlated, but not causal, variables such as ovulation rate.The models for each reproductive step were linked to predict the flock reproduction dynamics from ovulation to weaning. Each flock was considered to have 1000 breeding ewes with distribution in age and premating liveweight representative of the flock. Day of lambing was calculated based on the assumptions that the start of mating is d 103 the year for each flock, ewes were exposed to the ram for two cycles, the standard deviation in lamb birthweight was 0.80 kg for each birth rank, and the weaning day was d 340 of the year. Simulations were repeated 10,000 times to determine the expected role of perturbations in average premating ewe liveweight, average ovulation rate, variability of ovulation rate, embryo survival, lamb survival, standard deviation in lambing day and average ewe age on the kilograms of lamb liveweight at weaning per ewe exposed to the ram . Simulations were conducted by considering a fixed range of deviations from the mean for each flock. Changes in the distribution of ewe age were generated with a Gamma correction  and standard deviation  in weight for age class i for ages 2 to 9 are listed in Premating ewe liveweight was suitably described by a normal distribution. Premating ewe liveweight for each age class was also suitably described by a normal distribution. Premating ewe liveweight typically increases significantly from age 2 to 4 . The meai (ages 2 to 9 only).The ewe age distribution is listed in The distribution of the number of fetuses at mid-pregnancy for flock 2, which is representative of the distribution in the number of fetuses scanned in a typical New Zealand flock, is shown in W) on the number of ova for all flocks combined (P < 0.001), consistent with estimates from other studies (A) on the number of ova for all flocks combined (P < 0.001), consistent with other studies , standard deviation in gestation length (σg = 3 d) and the day of fertilization. The average day of lambing was d 262 of the year  and varied from d 247 to 275 between flocks. The mean and variance in the day of lambing will also be dependent on the number of cycles that ewes were exposed to the ram and variation between years in the date ewes were exposed to the ram, which were not recorded. Ewes were assumed to be exposed to the ram for two cycles.Day of lambing is dependent on the average gestation length (σyear = 3 d). A standard deviation in lambing day of 4.9 d is expected if all ewes lamb to the first cycle they were exposed to the ram . The relationship between estimated embryo survival probability from ovulation to scanning and the standard deviation in lambing day for each flock is shown in σyear = 0 d and is 6.5 d assuming σ year = 3 d (both assuming σg = 3 d and a 17-d cycle), which is consistent with minimum values in However, between year variability in lambing dates were relatively low within each flock (P < 0.001), consistent with other studies .The effect of ewe age on lamb survival as a function of the number of fetuses is shown in The effect of premating liveweight on the probability of lamb survival is also shown in P < 0.001) (P < 0.001). This effect was greatest for the lambs of lower birth rank (P < 0.001). There was significant between flock variability in both lamb growth rate and the effect of premating ewe liveweight on lamb growth rate (Lamb growth rate is significantly greater for lambs of lower birth rank (< 0.001) . There wwth rate .−1 growth advantage over twin lambs, consistent with other studies  that scanned one to five fetuses, respectively (only able to be estimated for flock 6). Ewes with four or five fetuses tended to lose BCS from mating to scanning. These changes in BCS from mating to scanning are therefore less than the 0.25 BCS decrease from premating to scanning in mature ewes observed by The decrease in BCS from scanning to weaning were 0.04 ± 0.02, 0.42 ± 0.007, 0.49 ± 0.01, 0.49 ± 0.02, 0.47 ± 0.08 in ewes with one, two, three, four, five fetuses scanned, respectively . There was therefore a very small decrease in BCS from scanning to weaning in ewes with a single fetus scanned and a 0.42 to 0.47 BCS decrease from scanning to weaning in ewes with multiple fetuses. This is similar to the 0.42 BCS decrease in mature ewes from premating to weaning observed by n = 53363, all ewes excluding ewe lambs) and 7.21 ± 0.07, 8.83 ± 0.16, 4.99 ± 0.15, and 5.33 ± 0.13 kg liveweight at mating in flocks 1, 6, 8, 9, respectively,  and was not able to be estimated for other flocks. These estimates are consistent with the difference of 7.3 kg per unit BCS observed by An increase in one BCS at mating equates on average to 8.45 ± 0.07 kg liveweight at mating  and liveweight (1.3 ± 0.2 kg) at mating the following year . This indicated that ewes that lost body condition in a given year (due to rearing multiple lambs) largely, but not completely, recovered lost body condition by mating the next year under the farm system for flock 6 .W,i) and standard deviation  in liveweight for ewe lambs (age class i = 1) are listed in Premating ewe lamb liveweight was be suitably described by a normal distribution . The meai (ages 1 to 9 only). Ewes in The ewe age distribution is listed in The distribution of the number of fetuses for flock 2 is shown in P < 0.0001). The effect of premating ewe lamb liveweight on ovulation rate was highly variable between flocks although ovulation rate typically increased with liveweight . Lamb survival was typically 81%, 71%, 43%, 38% for ewe lambs with one, two, three, four fetuses, respectively, although lamb survival varied by up to 10% between flocks  (P < 0.001). There was significant between flock variability in both the lamb growth rate and the effect of premating ewe lamb liveweight on lamb growth rate (Lamb growth rate was significantly greater for lambs of lower birth rank (< 0.001) . There wwth rate .BCS changes from mating to scanning were not able to be estimated for ewe lambs. BCS changed 0.02 ± 0.03, −0.30 ± 0.02, −0.39 ± 0.04, and 0.15 ± 0.15 BCS from scanning to weaning in ewe lambs with one, two, three, four fetuses, respectively (only able to be estimated for flock 6 for ewe lambs). BCS was unchanged from scanning to weaning in ewe lambs with a single lamb born and decreased 0.3 BCS from scanning to weaning in ewe lambs with multiple lambs born. This is similar to the 0.24 BCS decrease in ewe lambs from premating to weaning observed by n = 106, RMSE = 3.4 kg, all ewe lambs) and was not able to be estimated for other flocks.An increase in 1 BCS at mating equated on average to 4.58 ± 1.20 kg liveweight at mating  at mating the following year . This indicated that ewe lambs that lost body condition in a given year (due to rearing multiple lambs) largely, but not completely, recovered lost body condition by mating the next year under the farm system for flock 6 .We investigated the effect of average premating ewe liveweight, average ovulation rate, variability of ovulation rate, embryo survival, lamb survival, standard deviation in lambing day and average ewe age on the kilograms of lamb liveweight at weaning per ewe exposed to the ram , 2014.The calculated elasticities provide information on the expected change in flock performance and are therefore suitable for the strategic management of the flock. Although between year variation was not explicitly examined in this study, the between year variation in birthweight, litter size and lamb survival has an estimated coefficient of variation of 10% to 15 % . Within We investigated the effect of average premating ewe lamb liveweight, average ovulation rate, variability of ovulation rate, embryo survival, lamb survival, and standard deviation in lambing day on the kilograms of lamb liveweight at weaning per ewe lamb exposed to the ram  and for Reproductive loss was significantly greater in ewe lambs than mature ewes, although the relative difference was dependent on the stage of reproduction and was variable between flocks. Scanning rate was 55% lower for ewe lambs. Predicted ovulation rate was 25% lower for ewe lambs. There was a 30% relative decrease in the predicted embryo survival probability from ovulation to scanning for ewe lambs. There was a 10% relative decrease in lamb survival probability from birth to weaning for ewe lambs. Lamb growth was also 25% lower for ewe lambs. This is consistent with the 45% to 50% reduction in lambs to weaning , the 15%txab013_suppl_Supplementary_MaterialsClick here for additional data file.


Over one-third of older adults have a disabling hearing loss, with potentially severe implications for well-being. Hearing screening is not routine in primary care (PC) and patients are relied upon to report hearing concerns. We compared outcomes of three approaches to linking telephone-based screening with PC . This poster presents results of focus groups/interviews with providers and staff from participating clinics (n= 35), study enrollees who completed screening and were referred for diagnosis (n=14 ), and enrollees who did not complete screening (n=12). Results show that most patients had prior hearing concerns they had not reported to their PC. Patients forgot or were resistant to completing screening at home. Negative attitudes towards admitting hearing loss and using hearing aids were common; experiences of family and friends influenced many patient attitudes, both negative and positive. PC personnel wish to help, but are challenged by lack of time, space, and reimbursement for screening, and loathe to screen when specialty care and hearing aids are costly. Study results indicate that relying on patients to report hearing concerns is inadequate. Integration of hearing screening into PC would be helped by strengthening reimbursement for screening, specialty care, and hearing aids, and education of both providers and patients on other available treatments for hearing loss. Patients also require education on hearing aid technology. There is a need to address stigma associated with hearing loss, taking into consideration the influence of family and friends on attitudes.


Rhodobiaceae and Rhodobacteraceae families of Alphaproteobacteria, and unidentified members of the Candidate division Parcubacteria. Tolerant taxa, whose abundance increased significantly with higher TCS concentrations, include the families Erythrobacteraceae (Alphaproteobacteria), Flavobacteriaceae (Bacteroidetes), Bdellovibrionaceae (Deltaproteobacteria), several families of Gammaproteobacteria, and members of the Candidate phylum Gracilibacteria. Our results demonstrate the variability of TCS sensitivity among bacteria, and that TCS can change marine bacterial composition at concentrations that have been detected in the marine environment.The antibacterial agent Triclosan (TCS) is a ubiquitous environmental contaminant due to its widespread use. Sensitivity to TCS varies substantially among eu- and pro-karyotic species and its risk for the marine environment remains to be better elucidated. In particular, the effects that TCS causes on marine microbial communities are largely unknown. In this study we therefore used 16S amplicon rDNA sequencing to investigate TCS effects on the bacterial composition in marine periphyton communities that developed under long-term exposure to different TCS concentrations. Exposure to TCS resulted in clear changes in bacterial composition already at concentrations of 1 to 3.16 nM. We conclude that TCS affects the structure of the bacterial part of periphyton communities at concentrations that actually occur in the marine environment. Sensitive taxa, whose abundance decreased significantly with increasing TCS concentrations, include the  Triclosan -phenol, CAS 3380-34-5) is an antibacterial agent commonly used in personal care products (PCP), household cleaning products, textiles, and plastics. The annual usage of TCS and has been estimated at 300 tons in USA in 2005  reductase  glass slides mounted vertically in polyethylene holders. Prior to periphyton establishment, the discs were boiled for 10 min in concentrated nitric acid, rinsed in de-ionized water, and rinsed again in 70% ethanol. The seawater flow rate in the microcosms was 220 mL min−1 giving a mean residence time of approximately 90 min. TCS solutions were made in de-ionized water by adding TCS dissolved in acetone. These TCS solutions, containing 1 permille acetone (v/v), were pumped into the system at a dilution factor of 119 times, creating constant TCS nominal exposures of 0, 0.316, 1, 3.16, 10, 31.6, 100, 316, and 1000 nM. The same amount of de-ionized water, with the same amount of acetone, but without TCS was pumped into the control microcosms. The study thus had a concentration-response experimental design based on 13 replicates sampled from seven different concentrations of TCS , are reported in Eriksson et al. . In shorCS Table . TherefoCS Table .Table 1R2) per microcosm into filter-sterilized water from the respective microcosm. The biofilm material was pelleted by centrifugation at 15,000 g for 8 min, snap-frozen in liquid nitrogen, and stored at −80 °C. DNA extraction was performed using the FastDNA spin kit for soil  due to the high extraction yield obtained with this kit , samples were pooled in equimolar amounts. The pooled samples were sequenced at the SciLifeLab SNP/SEQ next generation sequencing facility  using Illumina MiSeq with a 2 × 300 bp chemistry following the protocols of the manufacturer.The raw sequence data were analyzed with a pipeline for de-multiplexing and sequence-pair assembly implemented in Python . Differentially abundant OTUs were identified using the DESeq2 R package. Two types of analyses were implemented: (i) pair-wise analysis between the untreated controls and the samples that were exposed to 3.16, 31.6, and 316 nM TCS, and (ii) regression analysis between OTU counts and TCS concentration using all 13 samples. The resulting p values were adjusted for multiple testing according to Benjamini-Hochbergs false discovery rate (FDR). OTUs with an FDR of less than 0.05 were considered statistically significant. In the pair-wise analysis, overrepresented taxa among the significant OTUs (FDR < 0.05) were tested using Fishers’ exact test at the phylum, class, order, and family levels. Venn diagrams were used to describe the overlap of the significantly different OTUs between concentrations. All analyses were performed in the statistical language R version 3.4 . This pattern is confirmed in the Principal Component Analysis (PCA)  Fig. . MoreoveCA) Fig.  and 2.FiThe relative abundance of a total of 164 OTUs was significantly affected (FDR < 0.05) by an exposure to 3.16, 31.6 or 316 nM TCS (Supplementary Table 3). The number of significantly affected OTUs increased with TCS concentration Fig. . 10 of tParcubacteria decreased substantially at a concentration as low as 1 nM , and 316 nM (p = 0.0027) TCS, respectively. The Candidate division Parcubacteria, also called OD1, is a diverse group of bacteria, suggested to constitute a superphylum -generating compound tellurite, and TCS is well known for its ROS-mediated toxic effects in various organisms , and at 316 nM the abundance of 61, 36, and 22% of the OTUs in Alteromonadaceae, Oceanospirillaceae, and Thiotrichaceae were significantly increased. These taxonomic groups were favored only at higher concentrations of TCS . Although Pseudomonas aeruginosa belongs to Pseudomonadales, i.e. a different gammaproteobacterial order, it is worth noting that P. aeruginosa is intrinsically resistant to TCS. This resistance is believed to originate from efflux pumps, but Zhu et al.  of TCS. It is, however, currently not known to what extent other Gammaproteobacteria carry a TCS-resistant fabV gene. As reviewed by Carey and McNamara . It is possible that ecological interactions changed at intermediate TCS concentrations, favoring these taxa. For example, Verrucomicrobia can be symbionts with ciliates  and found that the overall bacterial density and community composition of 16S in a marine biofilm was affected at a high TCS concentration of 1000 nM, but that the taxa Alphaproteobacteria and Gammaproteobacteria were affected already at 10 nM. In the present study, we identified Alphaproteobacterial taxa at lower taxonomic levels to be TCS-sensitive  are particularly sensitive to TCS while Gammaproteobacteria , Flavobacteria (primarily Flavobacteriaceae), the Candidate division Gracilibacteria, the deltaproteobacterial family Bdellovibrionaceae, and the alphaproteobacterial family Erythrobacteraceae are more tolerant to TCS exposure. The results show that TCS affects marine microbial communities at low nanomolar concentrations, which are close to those found in the marine environment (Pintado-Herrera et al. We identified clear changes in community composition at 10 nM TCS, but effects on specific taxa were seen already at 1–3.16 nM. Our results show that Candidate division The raw sequence data are deposited at NCBI under the BioProject accession number PRJNA320539, and with the SRA Experiment accession numbers SRX1744264–SRX1744266, SRX1744269–SRX1744273 and SRX1744275–SRX1744279.Supplementary materialSupplementary Table 3


We aimed at describing the possible role of mexiletine in CIDP management.A 44-year-old female affected by CIDP and MC, gained beneficial effects for CIDP symptoms  and relapses, after mexiletine intake (200 mg twice a day). The patient presented with detrimental effects after mexiletine drop out, with a worsening of CIDP symptoms.The patient reported a nearly complete remission of muscle stiffness and weakness up to 3 years since mexiletine intake. Then, she developed an allergic reaction with glottis edema, maybe related to mexiletine intake, as per emergency room doctors’ evaluation, who suggested withdrawing the drug.The patient significantly worsened after the medication drop out concerning both CIDP and MC symptoms.This is the first report on the association of CIDP and MC in the same patient. Such diseases may share some clinical symptoms related to a persistent sodium currents increase, which maybe due either to the over-expression of sodium channels following axonal damage due to demyelination or to the chloride channel genes mutations. This is the possible reason why mexiletine maybe promising to treat CIDP symptoms. The incidence of such peripheral demyelinating diseases is variable.–5 CIDP pathogenesis is likely to be immune mediated. CIDP onset is insidious.–10 Its clinical course is slowly progressive (over at least 2 months), with relapses, and includes symmetric weakness of both muscles around the hip and shoulder, as well as of the hands and feet , and sensory disturbances causing incoordination, numbness, tingling, or prickling sensations.–10 Other CIDP symptoms may include fatigue, burning sensation, pain, clumsiness, difficulty in swallowing, and double vision. Deep tendon reflexes are reduced or absent. Walking may be involved when responses to various sensory stimuli are seriously impaired.–10 The immune system firstly attacks the myelin sheaths of the PNS, inducing the typical segmental demyelination and then axonal degeneration. This gets slower nerve conduction velocity, reduces sensory and motor responses amplitude, and provokes conduction blocks at the neurophysiological studies. To date, oral glucocorticoids are considered the first line treatment, together with plasmapheresis and intravenous human immune globulin (IVHIG).,12Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an acquired immune mediated demyelinating disease of the peripheral nervous system (PNS), causing progressive loss of motor and sensory functions. MC is caused by mutations in the skeletal muscle chloride channel gene (CLCN1). There are 2 forms, the autosomal dominant (Thomsen MC) and the autosomal recessive (Becker MC) MC. The latter is associated to transient weakness, with recovery often induced by repetitive exercises. Mexiletine is the first choice treatment to improve myotonia-induced symptoms.Myotonia congenita (MC) belongs to the nondystrophic myotonias, which are a group of genetically and clinically heterogeneous ion channel muscular disorders, typically characterized by delayed muscle relaxation after voluntary contraction, stiffness, muscle hypertrophy, and cramping.Despite MC and CIDP are distinct clinical entities, they may share a common pathophysiological issue, that is, the channelopathy . Thus, a possible association between these 2 clinical conditions maybe not necessarily casual.Herein, we report on the first case of a woman affected by CIDP and MC, treated with mexiletine. The patient got detrimental effects on CIDP symptoms after mexiletine drop out. The patient provided her informed consent for publication of the case2In 2002, a 44-year-old female began to complain of symmetric proximal and distal weakness with difficulty in walking, mild sensory dysfunction with distal limb not-disabling paresthesia, and absent tendon reflexes in all extremities, which all gradually worsened over 2 months. She was then hospitalized elsewhere, and she underwent several investigations, including:(1)electroneuromyography (EMG) showing peripheral nerve demyelination: that is, conduction blocks, a reduction of nerve conduction velocities (NCV) <80% of lower limit of normal, with a discrete variability among nerves, dispersed compound muscle action potentials (CMAP), and prolonged distal latencies, as well as F-waves presence;(2)needle EMG showing no abnormal spontaneous activity, prolonged motor unit action potentials, and a reduced interference pattern), and(3)lumbar puncture, disclosing increased protein level (67 mg/dL) with a normal cell count (<5 mononuclear cells) in the cerebrospinal fluid. She was; therefore, diagnosed with CIDP, according to the EFNS/PNS diagnostic criteria for CIDP.She was treated with prednisone 100 mg/d p.o., which was withdrawn after the clinical improvement (about 2 weeks later). Within 1 month, she was at the level she had been before CIDP onset, but a residual mild numbness of fingertips and muscle stiffness were appreciable. There were no limitations in her daily life activities. The EMG pattern consisted of an improvement in CMAP amplitude, reduced distal latencies, improved NCV and F-wave latency, and conduction block disappearance. However, she was prescribed with methotrexate 25 mg per week, followed by folin administration the day after the injection. Over the years, she had 5 relapses (from 2002 to 2015), each of them characterized by mild distal limb paresthesia progressing to forearms and upper legs, diffuse weakness of arm and leg muscles, until she was not able to walk without assistance. Each relapse was treated with high-dose iv corticosteroids , and the methotrexate was supplemented with monthly iv pulse of cyclophosphamide for 6 months, with clinical beneficial effects each time. Indeed, she was at the level she had been before the relapse, within 10 days from iv corticosteroids, with only residual mild lower limbs muscle weakness and stiffness. Notably, since 2008, the patient began to use myorelaxant as needed to face the increasing muscle stiffness. No data on other EMG investigations were available.CLCN1 gene at our neuromuscular specialized center. Thus, the patient took part to a familial screening for MC, being provided with a neurophysiological examination following the Fournier guidelines for ion channel disorders. The exam confirmed a chloride muscle channelopathy, as in her daughter. The EMG pattern showed a reduced CMAP amplitude and NCV, and an increase in F-wave latency. The Fournier pattern II consisted of post-exercise myotonic potentials, a transient decline of CMAP amplitude after short exercise test after rest, and a reversion of the block of muscle excitability induced by repeating short exercise test. Unfortunately, the diagnosis was not supported by molecular analysis, since the patient refused to do it. In consideration of the residual symptoms (mild lower limbs muscle weakness and stiffness) and the probable MC diagnosis, the patient was then prescribed mexiletine (200 mg twice a day) and monitored by serial electrocardiograms to check the QT interval.In 2015, her daughter was diagnosed with recessive MC due to the Gly190Ser mutation in the The patient reported a nearly complete remission of muscle stiffness and weakness up to 2018. Also, she reported a CIDP remission, complaining only of mild numbness of fingertips. She did not practice any other specific investigation. Because the patient felt such a clinical improvement, she decided to suspend on her own the methotrexate assumption after 2 months from the mexiletine intake.Unfortunately, in 2018, she developed an allergic reaction with glottis edema, considered to be related to mexiletine by the emergency room doctors. Even though the cause of such allergy was neither tested nor confirmed, mexiletine was suspended. Some weeks later, the patient began to complain of walking problems with a progressive wide-based unsteady gait, mild distal lower limb paresthesia, muscle weakness, and fatigue. Such symptoms brought the patient to the Emergency Neurology Unit of our institute. On admission, the patient exhibited eyelid, lid-lag, handgrip and percussion myotonia, gait ataxia, difficulty in starting ambulation because of muscle stiffness, difficulty to walk on tiptoes and heels. Romberg sign was positive. There was a marked lower limb muscle weakness, more pronounced proximally. Deep tendon reflexes were decreased. She referred lower limb distal pain hypoesthesia and hypopallesthesia. EMG showed a pattern suggesting CIDP relapse ; also, myotonic discharges were appreciable in all examined muscles. A diagnosis of CIDP relapse associated with MC was thus confirmed. The patient was provided with IVHIG at the dose of 400 mg/kg/d for 5 days, with a quick improvement in gait, motor, and sensitive symptoms. Neurological examination after IVHIG showed eyelid, lid-lag, handgrip and percussion myotonia, difficulty in starting ambulation because of muscle stiffness, but with normal muscle strength and sensibility at the 4 limbs. Deep tendon reflexes were still decreased, together with a slight lower limb distal pain hypoesthesia and hypopallesthesia. IVHIG administration was repeated every week for 4 months. Also, azathioprine was started at the dosage of 100 mg/d.Actually, the patient complains only of the abovementioned MC-related symptoms. No CIDP relapses have yet occurred. The patient refused to reintroduce mexiletine because of the previous, even not confirmed, allergic reaction. The patient provided her written informed consent to study publication.3–17 Despite the different origin of these channelopathies, a pathophysiological correlation may exist; for example, the nerve pathology could negatively affect muscle membrane physiology with a change in channel function or expression. Anyway, the channelopathy causes an abnormal gain of channel function  or an abnormal loss of channel function , depending on whether the channelopathy leads to membrane hyper- or hypo-excitability. Thus, a possible association between these 2 clinical conditions maybe not necessarily casual.CIDP and MC are rare disorders of the PNS. To date, there is no report on their association in the same patient. In our case, MC diagnosis followed CIDP diagnosis, and it was made in the context of a genetic familial screening for ion channel disorders. Despite MC and CIDP are distinct clinical entities, they may share a common pathophysiological issue, that is, the channelopathy . The muscle chloride channelopathy represents the primary trait of MC. On the other hand, peripheral  nervous system channelopathies can be due to a demyelization process exposing channel structures at the level of nodes and paranodes. Generally speaking, voltage-gated sodium channel (Nav) dysfunction causes a failure in inactivating and conducting sustained inward Na+ fluxes, with consequential skeletal muscle hyperexcitability, or weakness in presence of high levels of extracellular K+. Voltage gated potassium channel (Kv) dysfunction is characterized by reduced K+ effluxes, thus prolonging the falling or repolarization phase of the action potential and inducing repetitive nerve firing (that causes excessive and unregulated neurotransmitter release at the neuromuscular junction). In addition, voltage gated chloride channel (Cl−) defects cause smaller Cl− influxes that in turn implies that smaller influxes of Na+ are needed to reach the threshold for action potentials, enhancing muscle excitability. Lastly, voltage gated calcium channel (Ca2+) dysfunction induces muscle weakness and paralysis due to a persistent membrane depolarization that inactivates the Nav channels.This association is supported by some common positive  and negative symptoms (muscle weakness) between such diseases. These symptoms can be caused by both the hyper- or hypo-excitability of the injured axons (secondary to the demyelization process) and by the muscle chloride channelopathy. that is, nodal axonal neurofascin-186 (NF-186), NF-155, contactin-1, NrCAM bind gliomedin, which are involved in Nav and Kv clustering, required for fast and saltatory action potential propagation.,24 Autoantibodies against NF-186, NF-155, gliomedin, and contactin-1 have been described in demyelinating inflammatory neuropathies.–29 Disruption of axon membrane induced by these antibodies leads to clustering defects of nodal-paranodal channels  and the exposure of the iuxtaparanodal Kv, with either positive or negative symptoms as a consequence.Channelopathy in MC is genetically defined, whereas channelopathy in CIDP is thought to be related to the damage induced by some antibodies involved in CIDP pathogenesis. In fact, physiologically, in order to maintain and organize the junctions at nodes of Ranvier, paranodes and juxtaparanodes there are the axoglial cell adhesion molecules,–32 This is what also occurs with the CLCN1 mutation of MC.Therefore, changes in nodal-paranodal channel functions in CIDP can lead to an enhanced excitability or to an inexcitability , depending on the prevailing channel dysfunction.11H17NO) is a sodium channel blocker belonging to the class IB of antiarrhythmic agents, which is structurally related to the anesthetic lidocaine. It has been approved in Italy for the treatment of myotonia on 2006. The bioavailability is of 90%, and peak plasma concentrations occur after 2 to 4 hours. The mean drug half-life is of about 11 hours. The pharmacokinetics is preserved up to a creatinine clearance of 10 mL/min. It is metabolized by the liver, predominantly. Mexiletine prolongs the refractory period by delaying the recovery from inactivation of Nav. Specifically, Mexiletine targets the Nav in a use-dependent and block voltage-dependent manner rather than blocking rest or tonically active Nav. Furthermore, it targets more downstream cell pathophysiology in the chloride channel. Therefore, Mexiletine is more active on muscle fibers subject to repeated discharges , thus reducing the delay in muscle relaxation and, eventually, muscle stiffness. Moreover, it is used for treating arrhythmias and chronic pain, consistently with its specific efficacy in blocking Nav that continues to release a sustained current rather than fully inactivating.–38Moreover, in our case, the possible correlation between CIDP and MC is also suggested by the positive effect of mexiletine administration on both MC and CIDP symptomatology. Indeed, the administration of mexiletine resulted in a nearly complete remission of muscle stiffness, cramping, and weakness. Mexiletine -2-propanamine - C,40 its effects on muscle weakness are less clear. We may speculate that mexiletine may have had challenged the abnormal nodal-paranodal channel activity (blockade of non-specific sodium channel) up to blocking glutamate-evoked activity in the dorsal horn of the spinal cord, beyond the well-known muscular effects. Such mechanism may confirm the pathophysiological correlation between CIDP and MC, and thus the drug maybe helpful in CIDP management.While the efficacy of mexiletine on muscle stiffness and cramping is intuitive and confirmed in different clinical trials, However, this issue remains unclear since the patient did not start over again the drug. Moreover, there is scarce evidence in literature on the possible immunomodulatory effect of mexiletine, mainly limited to a drug induced hypersensitivity syndrome.–46A secondary finding in our case consisted in the absence of CIDP relapses during the period of mexiletine intake, even after the methotrexate drop-out , and the abrupt CIDP relapse following mexiletine withdrawal. Altogether, these issues may configure the hypothesis of a sort of mechanism mimicking an immunomodulatory effect by part of mexiletine.–50 About that, experimental studies have shown that Nav are over-expressed beyond the nodal-paranodal sites in the growing and sprouting axons, as well as following functional denervation. Such a remodeling of Nav largely results in altered axonal and, consequently, muscle excitability.–53 In fact, previous studies have suggested that the increased excitability of motor axons in several diseases, including amyotrophic lateral sclerosis, spinal muscular atrophy, and peripheral neuropathies, can be due to an increase in persistent sodium currents, even autoimmune-based.–58In fact, it could be the case that a denervation hypersensitivity following lasting intake of mexiletine triggered the symptoms occurring after the discontinuation of the drug, rather than inducing an autoimmune reactivation. In turn, transmission of sensory signals and disinhibition or facilitation mechanisms are altered at the level of the spinal cord dorsal horn neurons.Last, it has to be acknowledged that peripheral neuropathy can alter the electrical properties of sensory nerves, which then leads to an imbalance between central excitatory and inhibitory signaling within inhibitory interneurons and descending control systems.v blockers, like mexiletine. About that, mexiletine may challenge muscle and nerve excitability, regardless of its origin  beyond its effectiveness on genetic muscle channelopathies. Obviously, such claim requires confirmation from clinical trials on CIDP patients, preferably without any electrophysiological evidence of muscle hyperexcitability.Our case suggests that CIDP and MC may share some clinical and pathophysiological features, with particular regard to channelopathy, being their association not necessarily casual. Moreover, we found that some CIDP-related symptoms could benefit from NaConceptualization: Simona Portaro, Rocco Salvatore Calabrò.Data curation: Simona Portaro, Fiammetta Biasini.Formal analysis: Antonino Naro, Fiammetta Biasini.Investigation: Simona Portaro, Fiammetta Biasini, Antonino Naro.Methodology: Simona Portaro, Fiammetta Biasini, Antonino Naro.Supervision: Rocco Salvatore Calabrò, Placido Bramanti.Validation: Rocco Salvatore Calabrò, Antonino Naro, Placido Bramanti.Visualization: Rocco Salvatore Calabrò, Placido Bramanti.Writing – original draft: Simona Portaro, Antonino Naro.Writing – review & editing: Simona Portaro, Antonino Naro, Rocco Salvatore Calabrò.


DFNA5) gene-related hearing loss. Here, we report a novel mutation (c.991-1G > C) in DFNA5, which co-segregated with late-onset ADNSHL in a Chinese family and was identified via exome sequencing and Sanger sequencing of DNA from peripheral blood of the family members. Further sequencing of cDNA derived from peripheral blood mRNA revealed that the c.991-1G >C mutation led to the skipping of exon 8, which is a known pathogenic mechanism for DFNA5-related hearing loss.Autosomal dominant non-syndromic hearing loss (ADNSHL) has a broad phenotypic spectrum which includes bilateral, symmetrical, and high-frequency sensorineural hearing loss, that eventually progresses into hearing loss at all frequencies. Several genetic variations have been identified as causal factors underlying deafness, autosomal dominant 5 ( DFNA5), which is known as GSDME in the context of cancer, is the fifth DFNA locus associated with ADNSHL. Numerous DFNA5-associated splice-site variations have been reported as pathogenic mutations for hearing loss. All these variations have a common effect of skipping exon 8 at the mRNA level, which results in an identical gain-of-function effect at the protein level. Here, we report a novel pathogenic DFNA5 splice-site variant, which was identified in a Chinese family via exome sequencing and validated using Sanger sequencing. Our findings further support the pathogenicity of variants affecting the splicing of DFNA5 exon 8.Genetic hearing loss is a common congenital sensory disorder worldwide. In approximately 50% of cases, hearing loss is caused by genetic factors, and 70% of people with hereditary hearing loss are classified as cases of non-syndromic hearing loss, which is not associated with other diseases . AutosomWe recruited three members of a Chinese family affected by late-onset ADNSHL, including the proband (II-1) and his parents . Thus, tAll the family members agreed to undergo clinical evaluations, including a complete medical history and detailed physical examination. Auditory evaluations included otoscopic examination, otoacoustic emission, and pure-tone audiometry (PTA). All three members were subjected to air-conduction PTA, in which the hearing thresholds were determined at six frequencies . The same frequencies were used for bone-conduction PTA in the proband (II-1) and his mother (I-2). The severity of the impairment was judged as mild (20–40 dB HL), moderate (41–70 dB HL), severe (71–95 dB HL), and profound (>95 dB HL) hearing loss.In the proband, the ADNSHL was bilateral and symmetric, varying from moderate to profound, whereas in the proband’s mother (I-2), the ADNSHL was profound .1, ClinVar database2, and standard variants of the American College of Medical Genetics and Genomics (ACMG)  were selected for the analysis.Peripheral blood samples were obtained from all the individuals with ethical approval. Genomic DNA was extracted from the blood samples using the TIANamp blood DNA kit (TIANGEN). The genomic DNA of the proband was subjected to exome sequencing. Targeted exon sequences and their flanking sequences were captured and enriched using an array-based hybridization chip , followed by HiSeq X10 (Illumina) sequencing. All autosomal and sex chromosome variants were annotated using the ANNOVAR tool. The pathogenicity of the variants was annotated using the Human Gene Mutation Databases (ACMG) . A serieDFNA5 (GenBank: AF073308) gene fragment was amplified and sequenced using a pair of human DFNA5-specific primers (forward 5′-TGTAGCCACCAAGGATTAGCAA-3′ and reverse 5′-AGGGCACTGACCAAGAAGTAGG-3′). Sanger sequencing for all family members was conducted on the ABI 3730 platform (Applied Biosystems) and confirmed the presence of the c.991-1G >C mutation in DFNA5 (NM_004403.3), which co-segregated with the disease phenotype  and exon 10 (forward 5′- GCACAGAGTCCATTCAGGGT-3′) and used to amplify a fragment of cDNA generated from peripheral blood mRNA. mRNA was extracted using the RNAprep Pure tissue kit (TIANGEN), and was reverse transcribed to cDNA using the RevertAid first-strand cDNA synthesis kit . Polymerase chain reaction (PCR) was conducted using the Phanta Max super-fidelity DNA polymerase (Vazyme Biotech). The amplified fragments were sequenced on the ABI 3730 platform. Agarose gel electrophoresis of the PCR products revealed fragments of 585 and 392 bp in the proband cDNA and a 585-bp fragment in the control (DFNA5 coding sequence and located upstream of the variant: forward 5′-CGCCTGGAAGATGTCACTCA-3′ (exon 7) and reverse 5′-ACTCTGTCTACCTGGACCCC-3′ (exon 6). The results of qPCR revealed that the relative expression of DFNA5 mRNA in the proband was 1.482-fold of that in the control (To investigate the potential effect of the c.991-1G >C mutation in  control . Sequenc control . Skippin control . qPCR wa control .DFNA5 mutations reported to date are splice-site variants leading to the skipping of exon 8 (DFNA5 mutation is bilateral, symmetrical, late-onset hearing loss, starting at high frequencies and ultimately progressing to involve all frequencies, with the severity ranging from moderate to profound. Consistently, the affected family members evaluated in this study showed these typical clinical symptoms. In addition to the previously reported c.991-2A >G mutation, which is located at the conventional AG/GT splice site (All f exon 8 . The comice site , we idenDFNA5 encodes a protein of 496 amino acids, which is expressed in the human cochlea (DFNA5 transcripts results in the translation of a truncated protein with apoptosis-inducing activity, owing to the loss of the protective C-terminal domain. In vitro, a cDNA construct with deleted exon 8 was shown to induce programmed cell death in both human and yeast cell lines (–/–Dfna5 and +/–Dfna5 knockout mice did not exhibit hearing loss, despite a divergent number of cochlear fourth-row outer hair cells ( cochlea . Skippin cochlea . The DFN cochlea . Skippinll lines . Howeverir cells . The priir cells .DFNA5 splicing variant, c.991-1G >C, in a Chinese family. This finding supports the pathogenicity of variants that affect the splicing of DFNA5 exon 8.In conclusion, we identified a novel liucx1716@163.com.The datasets for this article are not publicly available due to concerns regarding participant/patient anonymity. Requests to access the datasets should be directed to the C-XL, This study was approved by the Medicine Ethics Committee of the Shengjing Hospital of China Medical University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the proband and his parents for the publication of any potentially identifiable images or data included in this article.XC and YL conceived and designed the experiments. M-HL, YL, and C-XL helped with the patient’s workup and recruitment of the patient and his family members. XC, B-LJ, and M-HL performed the experiments and helped with genetic analysis. XC, B-LJ, and YL wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


BRAF mutation (23%), predominantly p.V600E (71%), and alterations in RB1 (47%), particularly deletion (57%). The frequencies and distributions of other genes, such as KRAS, APC, SMAD4, and PIK3CA, and microsatellite instability status were similar to those of ACs. These findings provide beneficial information for selecting therapeutic options, including targeted therapy, and a better understanding of the histogenesis of this tumour.Poorly differentiated neuroendocrine carcinoma (NEC) is a rare subtype of colorectal cancer (CRC). This study aimed to investigate clinicopathologic characteristics of colorectal NECs and elucidate genomic differences and similarities between colorectal NECs and colorectal adenocarcinomas (ACs). A total of 30 colorectal NECs were screened for frequently identified CRC oncogenic driver genes by targeted next-generation sequencing of 382 genes. The median age of the patients was 67 years . NECs occurred predominantly in the rectum (47%) and exhibited multiple adverse prognostic pathologic factors, including frequent lymphatic and vascular invasions, high rates of lymph node metastasis and distant metastasis and advanced TNM stage. The 1-, 3-, and 5-year overall survival rates of NEC patients were 46.7%, 36.4%, and 32.7%, respectively, with a median overall survival period of 11.5 months. In a molecular analysis, NECs showed high rates of  The majority of patients present metastatic disease at the time of diagnosis3. Colorectal NECs exhibit aggressive biologic behaviour with short-lived responses to therapy and inferior prognosis compared with typical CRCs5. Treatment strategies for colorectal NECs generally follow the chemotherapy regimens used for pulmonary NECs, consisting of platinum-based chemotherapy as the first-line therapy. Nonetheless, the prognosis of colorectal NECs remains dismal, with a low median survival of 5–11 months1.Poorly differentiated neuroendocrine carcinoma (NEC) in the colorectum is rare, accounting for approximately 0.6% of all colorectal carcinomas (CRCs)2), and a high Ki-67 index (> 20%)6. Morphologically, NECs are divided into small-cell or large-cell types, similar to their pulmonary counterparts. Recently, it has been recognized that there are a subset of well-differentiated neuroendocrine tumours with a high proliferation index 5. Although well-differentiated neuroendocrine tumours with a high proliferation index are classified as “grade 3 tumours” according to the current WHO classification system, they show different molecular alterations and decreased response to platinum-based therapy compared with small-cell or large-cell NECs5. Thus, poorly differentiated NEC is considered a different disease entity that is distinct from well-differentiated neuroendocrine tumours with a high proliferation index, WHO grade 3.According to the World Health Organization (WHO) classification of gastrointestinal neuroendocrine tumours, NECs are characterized by high-grade morphology, frequent necrosis, increased mitotic figures  including TP53, APC, KRAS, PIK3CA, BRAF etc10. BRAF mutation frequencies have been reported variably, ranging from 4 to 59%, as revealed by Sanger sequencing or targeted next-generation sequencing. In addition, KRAS mutations occur in 8% to 70% of cases, and the frequency of TP53 mutation reportedly ranges from 21 to 80%. In a recent series of 25 colorectal NECs, Shamir et al. found RB1 alterations in 14 tumours (56%), with TP53 being mutated in 12 (48%), similar to the frequencies of small-cell carcinoma of the lung11. In contrast, other frequently identified somatic mutations in colorectal ACs, such as mutations in BRAF, KRAS, and PIK3CA, but not in APC, were less common than in other studies10. Due to the rarity of this disease entity, the frequencies of identified mutations in colorectal NECs have been inconsistently described. Therefore, our study adds an additional 30 pure NEC cases to the previous literature, which helps to validate the prior molecular data of colorectal NECs.Several prior studies have investigated the genetics of colorectal NECs which have been reported to have frequent alterations in BRAF mutations, particularly p.V600E, are significantly more common in right-sided CRC (24.2% vs. 2.1%)12. Right-sided CRCs had higher rates of microsatellite instability and PIK3CA mutations and increased mutational burden, whereas mutations in APC and TP53 were enriched in left-sided CRC (81.9% vs. 63.6% and 64.6% vs. 34.8%). Considering that the different mutational rates of key oncogenic driver genes of CRCs depend on the tumour site, a specific analysis would involve comparison of identified mutations of colorectal NECs with those of colorectal ACs after tumour site matching.It is known that different frequencies and distributions of mutations in key oncogenes and tumour suppressors exist between right- and left-sided CRCs. In comparison analysis of frequently identified somatic mutations in right- and left-sided CRCs from the Cancer Genome Atlas dataset, The main purpose of this study was to better elucidate the clinicopathologic features and clinical outcome of colorectal NECs and genomic distinctions as well as similarities between these tumours and conventional ACs. We also investigated potential therapeutically targetable molecular alterations in colorectal NECs to optimize patient selection for new drugs and their combinations.2) and posttherapy specimens of colorectal NECs were excluded. Mixed adenoneuroendocrine carcinoma (MANEC) is a mixed malignant neoplasm with a neuroendocrine component combined with a glandular component. Each component should account for at least 30% of the tumor cell population6. To maximize DNA extraction of the pure component of NEC for molecular analysis, cases of MANEC were also excluded. Poorly differentiated NEC cases with more than 70% neuroendocrine components were selected for molecular and immunohistochemical analyses. Tumours were morphologically classified into small-cell or large-cell types according to the WHO classification criteria6. Tumours consisting of small to medium-sized cells with a high nuclear-cytoplasmic ratio, scant cytoplasm, and fusiform nuclei containing fine granular chromatin without prominent nucleoli were considered small-cell NEC. Large-cell NECs were considered those consisting of large polygonal cells with round nuclei, moderate amounts of cytoplasm, and sometimes prominent nucleoli. The mitotic index was obtained by evaluating the most mitotically active 2 mm2 area of the tumour. For a control group, initially, 120 surgical cases of colorectal AC after matching the tumour site were searched from the pathology report profile and reviewed. In cases of suspicious neuroendocrine differentiation within a tumour, immunohistochemistry for neuroendocrine markers was performed to confirm neuroendocrine differentiation. Ultimately, 100 surgical cases of colorectal ACs without neuroendocrine differentiation were selected as a control group. Histologic findings including mitotic count, depth of invasion, lymphatic invasion, vascular invasion, perineural invasion, regional lymph node metastasis, distant metastasis, and pathologic TNM staging were also evaluated. Tumours were staged according to the 8th edition of the American Joint Committee on Cancer (AJCC) TNM staging system for CRCs after the clinicopathologic features and radiologic findings had been re-reviewed13. Demographic and clinical information, including age, sex, underlying disease, type of surgery, tumour site, date of diagnosis, postoperative treatment, last follow-up status, date of death or last follow-up, were collected from a patient medical record review.The institutional review board of Asan Medical Center (AMC), Seoul, South Korea, approved this study. All methods were performed in accordance with relevant guidelines and regulations. We reviewed the records of patients diagnosed with poorly differentiated NECs and neuroendocrine tumours of WHO grade 3 in the colorectum from the Pathology Department at AMC. A total of thirty-five surgically resected cases were initially searched and reviewed. Neuroendocrine markers including synaptophysin and chromogranin and Ki-67 staining were performed for all thirty-five cases. Among them, well-differentiated neuroendocrine tumors of grade 3 , MSH2 , MSH6 , PMS2 , p53 , Rb1 , p16 , anti-Human papillomavirus . Anti-HPV is immunoreactive with paraffin sections of formalin-fixed tissues infected with HPV type 6, 11, 16, 18, 31, 33, 42, 51, 52, 56 and 58. An automated stainer  was used according to the manufacturer’s protocol. Immunohistochemical expression of each MMR protein was considered intact if nuclear staining of neoplastic cells was detected with internal control positivity in the non-neoplastic crypt epithelium. Expression of p53 was considered aberrant if there was diffuse and strong nuclear staining (more than 2/3 of cells) or complete loss of expression. Rb1, p16, and HPV were classified as immunoreactive if nuclear staining (Rb1 and HPV) or cytoplasmic staining (p16) in all neoplastic cells was observed. After reviewed p16 staining of colorectal NEC tumours, immunohistochemistry for HPV was performed on all p16 positive tumours to detect HPV infection.Microsatellite instability (MSI) analysis was performed on colorectal NEC tumours by multiplex polymerase chain reaction (PCR) with five quasimonomorphic mononucleotide repeat markers: BAT25, BAT26, D5S346, D17S250 and D2S123. Genomic DNA was isolated from paraffin-embedded tumour tissues using a QIAamp DNA Mini Kit . Each primer was end-labelled with one of the following fluorescent markers: FAM, HEX, or NED. An ABI Prism 3130 Genetic Analyzer  was used to analyse the products, and allelic sizes were estimated by Genemapper 4.1 . Tumours with allelic size variation in two or more of the microsatellite markers were deemed to be MSI-high, whereas tumours with allelic variations in one of the microsatellites were classified as MSI-low. If there were no allelic size variations, all microsatellites were considered microsatellite-stable (MSS).14. Briefly, the cancer-related genes evaluated included ABL1, AKT1, AKT2, ALK, APC, ARID1A, BRAF, CDH1, CSF1R, CTNNB1, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CDKN2C, EGFR, ERBB2, ERBB3, ERBB4, FBXW7, GNAS, HRAS, NRAS, PIK3CA, PIK3CB, PIK3CD, and SMAD4, among others. Overall, the panel covered 823,971 bp. A DNA library was prepared as described in our previous report using the S1 method14. Each library was constructed with sample-specific barcodes six-bp in size and quantified using Qubit Kit. Eight libraries were pooled for hybrid capture using the Agilent SureSelect XT custom kit . The enriched target concentration was measured by quantitative polymerase chain reaction . DNS libraries that passed quality checks were sequenced using MiSeq. Sequenced reads were mapped to the human reference genome (NCBI build 37) using Burrows-Wheeler Aligner (version 0.5.9) with default options. PCR duplicates were removed using the Picard tool. Then, de-duplicated reads were realigned at known indel positions using GATK IndelRealigner, and base quality was recalibrated with GATK Table Recalibration15. Somatic mutations of single-nucleotide variants and short indels were called in tumour tissue with matched normal tissue using MuTect (1.1.7) and SomaticIndelocator in GATK17. Germline variants from somatic variant candidates were filtered out using the common dbsnp database , the Korean Reference Genome database18 and an in-house panel of normal variants20. Filtered somatic variants were annotated with Variant Effect Predictor (v79) and then converted to MAF files using vcf2maf (v1.612)21. False-positive variants were manually curated using Integrative Genomic Viewer22.After a review of matched haematoxylin and eosin-stained slides, formalin-fixed and paraffin-embedded (FFPE) tissue blocks containing adequate tumour cellularity (> 70%) were selected. The area with a pure neuroendocrine component of NEC was carefully circled by a molecular pathologist (C.S.). Two to five sections (6 µm thick) from the circled area in each FFPE tissue were obtained. After deparaffinization with xylene and ethanol, gDNA was isolated using NEXprep FFPE Tissue Kit . Quantification was performed using Qubit dsDNA HS Assay Kit . Targeted next-generation sequencing was performed using MiSeq  with the OncoPanel AMC version 3.0 panel . This panel covers approximately 1.2 Mb with 33,524 probes targeting a total of 382 genes, including the entire exons of 199 genes, 184 hot spots, and partial introns for eight genes often rearranged in cancer, as previously describedt-test or the Mann–Whitney U-test. Overall survival curves were constructed using the Kaplan–Meier method and compared with the log-rank test. Cox proportional hazard models were employed to estimate the combined influence of clinicopathologic variables on survival. A p-value < 0.05 was considered statistically significant.Pearson's chi-square test or Fisher's exact test was applied to evaluate correlations between clinicopathologic variables and frequently mutated genes. Continuous variables were analysed using Student’s Informed consent was obtained from all study participants. This study was approved by the institutional review boards at Asan Medical Center, Seoul, South Korea. The study was conducted in accordance with the Declaration of Helsinki.2 was 62.5, ranging from 12 to 141/2mm2. The proliferation index assessed by Ki-67 was high, with a median percentage of 75% . The coexisting component within NEC tumours was predominantly adenocarcinoma (23%) or tubular adenoma (10%). Metastatic disease was noted in 11 (36.7%) patients at diagnosis, with the liver being the most common organ involved (54.5%), followed by distant lymph nodes (27.3%) and the ovary or peritoneum (18.2%). In the comparison analysis of colorectal NECs and ACs, patients with colorectal NECs were found to be slightly older than those with AC (mean age: 67 years vs. 62 years). Although there was no significant difference in tumour size or T category between the two groups, colorectal NECs tended to more frequently be associated with lymphatic invasion , vascular invasion , lymph node metastasis , and distant metastasis , leading to a more advanced disease stage  compared with those of 100 conventional ACs.A comparison analysis of the clinicopathologic features of colorectal NECs and ACs is summarized in Table The immunohistochemistry and microsatellite instability results are provided in Table BRAF mutations were detected in 7/30 (23%) tumours. The p.V600E BRAF mutations was observed in 5/7 (71%) tumours. Two (29%) tumours harboured BRAF mutations p.D594G and p.K601E. Activating RAS mutations (16 KRAS and 1 HRAS mutation) were identified in 16/30 (53%) tumours, predominantly in codons 12 and 13. GNAS mutations p.R201C and p.R201H were detected in three tumours (10%). TP53 mutations were identified in 13/30 (43%) tumours but without consistently mutated hot spots. Fourteen (47%) NEC tumours showed distinct alterations in RB1, accompanied by loss of Rb1 and overexpression of p16 by immunohistochemistry. RB1 alterations in 8/30 (27%) tumours presented predominantly as homozygous biallelic deletions, and activating RB1 mutations were observed in 6 (20%) tumours. Moreover, alterations of RB1 co-occurred with TP53 mutations in 4/13 (31%) tumours and frequently occurred alone. Among three tumours expressed Rb1 and p16, one Rb1 positive tumour with diffuse labelling for p16 was found to have a CDKN2A mutation by NGS. However, two Rb1 positive tumours with patchy labelling for p16 did not show relevant molecular alterations with Rb1/p16 pathway. Additionally, mutations in various genes were occasionally detected, including TET2, NOTCH1, NKX2-1, CHECK2, ATM, TS1, TS2, PTCH2, RHOA, DOT1L, ZNRF3, and ANTXR2. The majority of these mutations are of unknown functional consequence.All thirty NEC samples with more than 70% tumour cellularity were examined using the 382-gene panel. The somatic mutations and gene alterations identified in the 30 NEC tumours are detailed in Fig. BRAF mutations , particularly p.V600E (71%). The frequencies of identified KRAS mutations were similar in both groups (53% (NEC) vs. 53% (AC); p = 1.0). Although the frequencies of TP53 and APC mutations in the NEC group were slightly higher than those in the AC group , PIK3CA mutations were less frequently identified in the NEC group . Overall, there was no statistically significant difference in the frequencies of mutations in TP53, APC, or other genes, including SMAD4, PIK3CA, PTEN, and FBXW7, which have been reported to be mutated in colorectal ACs by next-generation sequencing.Molecular data for one hundred colorectal ACs matched by tumour site were analysed for comparison. The results of the comparative analysis of gene mutations in NECs and ACs are depicted in Fig. p = 0.031), lymph node metastasis (p = 0.02), and distant metastasis at diagnosis (M category) (p < 0.001) to be significantly associated with shorter OS . In multivariate analysis, distant metastasis at diagnosis (M category) was the only independent prognostic factor related to OS  patients had expired due to colorectal NEC, and 9 (30%) patients were alive with no evidence of recurrence. The 1-, 3-, and 5-year overall survival (OS) rates for all 30 patients were 46.7%, 36.4%, and 32.7%, respectively, with a median follow-up of 11.5 months (range 1 to 132 months). Kaplan–Meier survival analysis showed a high T category (p < 0.001).We also compared the OS rates of patients with colorectal NEC and those with 100 colorectal adenocarcinomas Fig. . Among 11. The clinicopathologic features and prognosis of colorectal NECs have been described in a few relatively large series. With a median OS of 11.5 months, the findings demonstrate that poorly differentiated colorectal NEC has an aggressive biologic behaviour with a poor clinical outcome, in line with prior studies. A retrospective study of 100 colorectal NECs analysed at M.D. Anderson Cancer Center reported a median age at diagnosis of 55 years (range 33–88 years), with 51% of the patients being men23. The majority of poorly differentiated NECs had a small-cell morphology (89%) rather than a large-cell morphology (8%), and metastatic disease was noted in 64 (64%) patients at diagnosis. In another retrospective analysis of data from the National Cancer Database (2004–2015) consisting of 1208 poorly differentiated colorectal NECs, the median age at diagnosis was 65 years, and 50% of patients were male3. A small-cell morphology was slightly more frequent than a large-cell morphology (54% vs. 46%). Lymphovascular invasion was observed in 25% of NEC cases, and approximately 55% of patients had advanced disease (III/IV) at diagnosis. In addition, Takaziwa et al. reported that patients with colorectal NECs were diagnosed at older age  and with a male predominance (2:1)7. Among 25 colorectal NECs, a large-cell morphology was more commonly observed than was a small-cell type (64% vs. 36%). NEC tumours showed high rates of lymph node metastasis (82%) and distant metastasis (44%) and were at a more advanced stage . In the present series, the patients were diagnosed at an older age (mean age: 67 years), with a male predominance , and among the 30 NEC tumours, a large-cell morphology was more frequently observed than a small-cell morphology (60% vs. 40%). Compared with typical ACs, NECs revealed multiple adverse prognostic pathologic factors, including frequent lymphatic invasion (97%), vascular invasion (77%), lymph node metastasis (80%), and distant metastasis at diagnosis (70%), which are associated with advanced TNM stage . Interestingly, based on the data from the Japanese and current series, patients with colorectal NECs in East Asian countries tend to have more adverse clinicopathologic factors related to advanced TNM staging than do patients in Western countries. However, due to the rarity of this disease entity, discrepancy in the analysed clinicopathologic findings of colorectal NECs depending on racial/geographic differences or observer variations is unclear.Poorly differentiated colorectal NEC is a rare malignant neoplasm accounted for only 0.6% of all CRCs25. According to poorly differentiated colorectal NEC data from the National Cancer Database (2004–2015), the median OS for patients who underwent surgical resection was 10.5 months compared with 6.9 months for patients who did not undergo surgery (p < 0.001)3. In our series, the median OS was 11.5 months; the 3-year survival was 36.4%, and the 5-year survival was 32.7%. The patients in our series with surgically resected colorectal NECs had a better OS than those who did not undergo surgery in the data from the National Cancer Database (11.5 months vs. 6.9 months). Regardless, it is uncertain whether this correlation is due to selection bias or true therapeutic effects. Thus, further studies about the survival benefit of surgical resection in treating colorectal NECs are needed.Unlike colorectal ACs, surgery alone is rarely curative for poorly differentiated NECs, and the survival benefit from surgery is controversial in prior studiesBRAF mutations, predominantly in exon 15, namely, p.V600E26. BRAF mutation is known to be prevalent in tumours of the proximal colon and tumours with poor differentiation (grade 3 or 4), mucinous histology, and high microsatellite instability26. However, the frequencies of BRAF mutations in poorly differentiated colorectal NECs are controversial (Table BRAF mutations in the form of p.V600E (80%), p.G469A (10%), or p.R671Q (10%)27. Olevian et al. found that seventeen (59%) of twenty-nine NECs had BRAF mutations in the form of p.V600E (88%), p.F595L (6%), or p.K601N (6%) by Sanger sequencing8. Additionally, Jesinghaus et al. reported different frequencies of identified BRAF mutations between MANECs and pure NECs, with an average of 33.3% of samples showing BRAF mutations9. In their study, colorectal MANECs more frequently had BRAF mutations  than pure NECs . In our series, seven (23%) of thirty pure NECs harboured BRAF mutations in the form of p.V600E (71%), p.D694G (14%), or p.K601E (14%). For comparison, six (6%) of a hundred conventional colorectal ACs harboured BRAF mutations in the form of p.V600E (83%) or p.G466V (17%). Because different frequencies of BRAF mutations in CRC have been reported depending on tumour location, we extracted the molecular data of conventional colorectal ACs matched by tumour location with the colorectal NEC group. Based on our data, BRAF mutations were more frequently identified in poorly differentiated colorectal NECs than in conventional colorectal ACs . Several case series of BRAF-targeted therapy have shown promising responses in patients with BRAF-mutated CRCs, including poorly differentiated NECs28. Considering that colorectal NECs harbour more BRAF mutations than do other types of CRC, it is critical to perform molecular testing for BRAF in patients with colorectal NEC to optimize patient selection for promising therapeutic options, such as BRAF inhibitors.Approximately 8% of CRCs harbour RB1 is a critical negative regulator of the Rb1/p16 pathway, which controls the G1 checkpoint of the cell cycle29. Inactivating mutations or homozygous deletions in RB1 have been reported in neuroendocrine neoplasms of the lung, gastrointestinal tract, and prostate32. Furthermore, a comprehensive whole-genome sequencing study of 110 pulmonary small-cell carcinomas found near-universal biallelic inactivation of RB1 via mutations, deletions, and complex rearrangements31. Nonetheless, there are contradicting data on the frequency of molecular alterations in RB1 in colorectal NECs. Only one (3.7%) patient with heterozygous RB1 loss was reported in a previous molecular analysis of 27 colorectal MANECs and NECs by targeted next-generation sequencing9. In contrast, a recent molecular analysis of 24 colorectal NECs and MANECs revealed that fourteen (58%) tumours showed biallelic alterations in RB1 with next-generation sequencing using a 479-gene panel11. Similarly, in our series, fourteen (47%) pure NECs displayed genomic alterations in RB1, including both mutations and deep deletions, with the latter being more prevalent (57%) than the former (43%). The tumours with RB1 alterations also exhibited aberrant expression of Rb1 and p16 by immunohistochemistry. Genomic alterations in RB1 accompanied by loss of Rb1 and overexpression of p16, representing deregulation of the Rb1-p16 pathway, are commonly observed in colorectal NECs. Prior and our data support that deregulation of the Rb1-p16 pathway, as confirmed by immunohistochemistry and next-generation sequencing, plays a critical role in the histogenesis of colorectal NECs, as it does in small-cell lung cancer. Unlike a study result by Shamir et al., we failed to find an association between p16 positive colorectal NECs and HPV infection. The difference of HPV infection status in colorectal NEC tumours between two studies may be caused by selection bias, presence of NEC tumours coinfected with HPV, or others. Further studies would be needed for investigating the role of HPV infection in the histogenesis of a subset of colorectal NEC tumours.TP53, KRAS, APC, PIK3CA, and PTEN, have been inconsistently reported in prior studies (Table 11. Two recent studies demonstrated relatively high mutation rates of TP53, KRAS, and PIK3CA, roughly similar to those of our molecular data11. However, two other studies showed relatively lower mutation rates for these genes10. These studies did not compare these mutated genes of colorectal NECs with those of colorectal ACs, and it remains unclear whether there are significant differences in these mutated genes in colorectal NECs. Thus, the present study compared these mutations of colorectal NECs with those of colorectal ACs after tumour site matching and detected no significant differences in the frequencies of TP53, KRAS, APC, PIK3CA, PTEN, GNAS, and SMAD4 mutations between 30 NEC tumours and 100 AC tumours. The almost parallel frequencies and distributions of the main oncogenic gene mutations, except for BRAF, identified between the NEC and AC groups indicate that colorectal NECs are genetically similar to colorectal ACs, suggesting that colorectal NECs arise from the same origin as colorectal ACs, with intestinal glands likely serving as the primary origin.The frequencies of other oncogenic driver genes commonly identified in CRC, such as es Table 7–11. TwoBRAF mutations, predominantly p.V600E, were more frequently identified in poorly differentiated NECs than in conventional ACs. Additionally, almost exclusive deregulation of the Rb1/p16 pathway was revealed by immunohistochemistry and next-generation sequencing. Frequencies and distributions of the main mutated oncogenic driver genes, except BRAF, and the microsatellite instability status were similar in NECs and ACs. These findings provide beneficial information for the use of potential therapeutics such as BRAF inhibitors and a better understanding of the histogenesis of this tumour.In summary, our study demonstrates that colorectal NECs display an aggressive biologic behaviour with multiple adverse clinicopathologic prognostic factors and a poor clinical outcome. In molecular analysis,