It was hypothesized that regional pancreatoduodenectomy (RPD), in which soft tissue around the PV is resected en bloc with its surrounding soft tissue. The extent of lymphadenectomy was similar between the procedures.This retrospective study included consecutive patients who underwent PD with PVR between January 2005 and December 2016 in a single high‐volume centre. In standard PD (SPD) with PVR, the PV was skeletonized and the surrounding soft tissue dissected. In RPD, the retropancreatic segment of the PV was resected versus 38 per cent in those having RPD; P = 0·014), and consequently they received neoadjuvant therapy less often . R0 resection was achieved in 73 patients (80 per cent) in the RPD group, compared with 117 (66·1 per cent) of those in the SPD group (P = 0·016), although perioperative outcomes were comparable between the groups. Median recurrence‐free (RFS) and overall (OS) survival were 17 and 32 months respectively in patients who had RPD, compared with 11 and 21 months in those who had SPD .A total of 268 patients were included . Tumours were more often resectable in patients undergoing SPD  procedure was employed to enhance oncological clearance around the mesenteric vein in patients with locally advanced pancreatic cancer. This provided better local control with longer overall survival than a standard pancreatoduodenectomy procedure.Regional pancreatectomy associated with increased R0‐rate and survival Complete resection is considered a cornerstone in curative therapy for pancreatic cancer, but sufficient margin clearance during pancreatoduodenectomy (PD) for pancreatic ductal adenocarcinoma (PDAC) has remained challenging because of the proximity to mesenteric vessels that must be preserved: the portal vein (PV), superior mesenteric vein (SMV) and artery (SMA). Although concomitant PV/SMV resection has been adopted widely and has improved the resectability of PDAC with suspected PV invasion, the positive resection margin rate remains high, ranging from 22 to 63 per centen bloc with the surrounding soft tissue (type I), in contrast to conventional PD with PV resection (PVR), in which the resected vessel is skeletonized except for the portion with tumour invasion in order to minimize the length of PV to be resectedIn 1973, Fortner and colleaguesThis concept was adopted into the authors' contemporary pancreatoduodenectomy with the SMA‐first approach, and a new surgical procedure was designed for PD. The new RPD approach is simpler and less radical than the original RP, involving no total pancreatectomy, no arterial resection and no extended lymphadenectomy. This study aimed to describe RPD and address its short‐ and long‐term outcomes compared with those for standard PD (SPD) with PVR in patients with advancedPDAC.The Cancer Institute Hospital Institutional Review Board approved the study. A retrospective review was undertaken of the medical records of consecutive patients who had PD for pancreatic adenocarcinoma from January 2005 to December 2016, and patients who underwent PD with PVR were included.The authors' approach to the management of pancreatic cancer has been described previouslyversus RPD) was chosen at the surgeon's discretion. The technical detail of the authors' standard PD (supracolic anterior artery‐first approach) has been described previouslyThe indication for PVR was determined based on preoperative CT findings: when direct contact of the tumour to the PV was suspected, PVR was planned. The type of resection  is ligated and divided before division of the pancreas along the line above or to the left of the PV/SMV. The soft tissue around the retropancreatic PV/SMV is then dissected and the PV/SMV skeletonized to minimize the length of vessel resection. The PMSC is preserved when possible, and resected only when in extensive contact with tumour.Fig. In RPD, the retropancreatic segment of the PV/SMV/PMSC and surrounding soft tissue are left untouched, and the pancreas is divided along the line above the SMA Fig. . The PV VideoThe first RPD was performed by the authors' group in 2008, and subsequently both procedures (RPD and SPD) have been performed according to surgeon preference. A representative video of RPD is provided as The pathologist handled all resected specimens according to the standard procedure; margin status was reported separately for the pancreatic stump, bile duct stump and dissected peripancreatic tissue. When the dissected peripancreatic tissue margin was positive, the location was described in detail. The orientation for each margin in the resected specimens was presented and verified at a weekly hepatopancreatobiliary pathology conference attended by surgeons and pathologists.A microscopically positive margin (R1) was defined by the presence of cancer cell in any margin, and R0 was reported when no cancer cells were detected in the margins, regardless of the distance between the tumour and the closed margin.Recurrence of the disease after resection was judged primarily by CT with intravenous contrast every 3–6 months after surgery, supplemented selectively by MRI and PET. Patients were usually prescribed systemic chemotherapy without histological confirmation by biopsy when CT and other clinical findings indicated disease recurrence.Local recurrence was defined as recurrence near the previous surgical bed or in the regional lymph nodes, and distant recurrence was defined as recurrence in the distant organs, including liver, lung and peritoneum.Data were retrieved retrospectively and analysed from medical records, including surgical and pathology reports, as follows: patient demographics; preoperative tumour characteristics including size and resectability based on NCCN guidelinesRecurrence‐free (RFS) and overall (OS) survival were determined from the time of resection to the time of detection of first recurrence and death or last follow‐up respectively. The dates for detection of initial local recurrence and distant recurrence were recorded separately.U test, and categorical variables are compared with the χ2 or Fisher's exact test. Survival curves were constructed using Kaplan–Meier methods and compared with the log rank test. Potential prognostic variables were evaluated by multivariable analysis using Cox regression. Two‐tailed P < 0·050 was considered statistically significant.Statistical analyses were performed using SPSS® version 24.0 . Continuous variables are expressed as median (range) values and compared with the Mann–Whitney en bloc PVR and were included in the study. Ninety‐one patients (34·0 per cent) underwent RPD and the rest had SPD with PVR. The demographics of the cohort patients and the tumour characteristics are summarized in Tableversus 39·5 per cent respectively, P = 0·014). As a result, more patients in the RPD group received neoadjuvant chemotherapy than those in the SPD group . Most patients did not receive neoadjuvant therapy as routine use of preoperative chemotherapy for borderline resectable tumours was initiated in 2015.Of 431 patients with PDAC who underwent PD during the study period, 268 (62·2 per cent) had Tableversus 3 cm respectively). Postoperative morbidity of Clavien–Dindo grade IIIa or above was similar regardless of procedure, and only two patients died from surgical complications, following SPD.versus 33·9 per cent respectively, P = 0·016; positive medial margin rate: 13 versus 28·2 per cent, P = 0·006) (TableP = 0·081).In terms of oncological outcomes, RPD was significantly less likely than SPD to result in positive resection margins, despite the similar median size of resected tumours .The median duration of follow‐up was 24 (range 1–163) months across the entire cohort and 38 (16–163) months in survivors. During follow‐up, 23 patients (25 per cent) who had RPD developed documented local recurrence, compared with 67 (37·9 per cent) who underwent SPD with PVR .TableUnivariable and multivariable analyses of predictive variables for RFS and OS following resection are presented in en bloc with surrounding soft tissue, and SPD in which the PV was skeletonized and resected only at the site of tumour invasion. RPD was safe and feasible with comparable surgical morbidity to SPD. Although there has been an ongoing debate about the role of radical surgical procedures for PDAC, this is the largest series among the few studies suggesting improved long‐term outcomes for RPD compared with SPD for patients with PDAC with PV invasion.This study from a high‐volume cancer centre compared two surgical approaches for PDAC with PV invasion: RPD in which retropancreatic segments of the PV, SMV and SpV were resected P = 0·03; incomplete resection: P < 0·001). Thus, PVR alone would add significant complexity and morbidity to the already complex pancreatic resection, and the surgical indication therefore needs to be selected carefully based on the surgeon's experience and skills following precise evaluation of the disease extent by preoperative imaging. The present authors perform PVR only when there is evidence of direct contact between the tumour and the PV on preoperative CT. Indeed, more than 60 per cent of the present PD procedures for PDAC were done with concomitant PVR. However, when the tumour to SMA contact exceeds 180°, the authors consider the tumour unresectable and no pancreatic resection is planned unless the tumour shrinks significantly with chemotherapy.It is important to acknowledge that this study is not a simple comparison of radical and non‐radical surgical procedures. Although the surgical safety of concomitant PVR during PD has been shown in many series, a meta‐analysis by Giovinazzo and co‐workersen bloc the right side of the periarterial neural plexus around the SMA when perioperative CT showed tumour to SMA contact of less than 180°According to the original Fortner reportAlthough lymph node metastasis was a significant prognostic factor in many studieset al.It should be noted that both SPD and RPD described in this report are variants of the ‘artery‐first’ PD in which SMA dissection with IPDA division is completed before the pancreatic division. As shown previouslyen bloc with the PV/SMV is the most critical oncological element for RPD. The first reason for this is the poor accuracy in assessing the extent of tumour infiltration around the PV. Wang et al.en bloc with retroperitoneal fat showed microscopic retroperitoneal fat infiltration.The authors believe that complete clearance of the entire retropancreatic soft tissue (fat) versus SPD with PVR was deferred to the surgeon based on their experience and preference. The initial RPD was performed in 2008, and the number of RPD procedures increased slowly, probably because of the learning curve for each surgeon; in recent years, particularly after 2015 when the new neoadjuvant therapy protocol was introduced, RPD has been performed more commonly than SPD with PVR. Furthermore, although no definitive selection criteria were set for the two procedures, surgeons preferred RPD over SPD for some advanced tumours, as R0 resection was not possible otherwise. As a result, tumour characteristics and use of neoadjuvant therapy were different in the two groups, and thus the comparison of outcomes needs to be interpreted with caution. Nevertheless, because more patients with complex tumours, such as borderline resectable or unresectable locally advanced disease, were included in the RPD group than in the SPD group, the present observations for comparable perioperative outcomes across the groups were sufficient to prove the safety and feasibility of RPD. As most of the cohort patients were treated before 2015, when the management protocol was changed for advanced PDAC, perioperative management in this cohort was somewhat outdated. In current practice, patients with borderline resectable PDAC would receive neoadjuvant therapy with multiagent systemic chemotherapy before definitive resection. Furthermore, the reported margin status in this study was also based on the traditional R definition, in which R0 was not defined by the margin length (more than 1 mm)Despite the scale and novelty of this study, there were some limitations. First, the study design of a retrospective single institutional review incurs inherited selection bias. Although the plan for PVR was always made before surgery based on CT findings, the choice of RPD en bloc retropancreatic PV/SMV resection with surrounding soft tissue is safe and feasible, and might also improve the long‐term outcomes. A prospective randomized trial to evaluate the role of RPD is warranted in the era of modern multidisciplinary management strategy for PDAC.RPD characterized by Video S1 Representative video of the regional pancreatoduodenectomy procedureClick here for additional data file.


To develop a post‐traumatic osteoarthritic model of hip following fracture of acetabulum in rabbit for revealing biochemical mechanism of post‐traumatic osteoarthritis.A total of 36 mature male New Zealand white rabbits were equally divided into sham group (n = 12), non‐ORIF group (n = 12), and open reduction and internal fixation (ORIF) group (n = 12). Except for the sham group, rabbits had survival surgeries to create acetabular fractures of dorsal wall for simulating dashboard impaction mechanism. The ORIF group received open reduction and internal fixation, while fractures in the non‐ORIF group were left as displaced but transverse fracture and dislocation was reduced. Besides intraoperative appearance and postoperative recovery, macroscopic and radiographic characteristics of the hips were recorded and assessed by a radiographic scoring scale at 3 weeks, 6 weeks, and 6 months, respectively.P = 0.011, 0.011, 0.015, respectively, <0.0167). Although the scores of the ORIF group showed apparent improvements (increased from 0.67 at 3 weeks to 2.00 at 6 months), there was no significant difference between the two modeled groups at all three time points.Out of 24 modeled acetabula, 21 (87.5%) were pure dorsal wall fractures as proposed and the remaining three were associated fractures  accompanied by dorsal dislocation or not. All hips were stable, and no sciatic nerve injury was observed. One rabbit in the ORIF group died of deep infection 4 days after surgery. Rabbits in the sham and ORIF groups returned to normal gait in 2 weeks, but animals in the non‐ORIF group suffered from limping and restricted movement. As the time progressed, the hips in the non‐ORIF group experienced progressive and severe degeneration which exhibited dramatically malformed and hypertrophic joints at 6 months, but the ORIF group maintained much better morphological structure. Corresponding to morphological changes, the average radiographic scores of the non‐ORIF group increased from 1.25 at 3 weeks to 2.75 at 6 months and showed statistically significant difference when compared to the sham group at all three time points  underwent a sham surgical procedure, while the other two groups underwent modeled procedures, which attempted to simulate dashboard impaction mechanism and create an intra‐articular dorsal wall fracture, with (the ORIF group) or without internal fixation (the non‐ORIF group). Even as early as 1964, Letounel and Judet first recommended open reduction and internal fixation (ORIF) surgery for acute acetabular fractures which were incongruent or unstable in order to get a better long‐time resultAs the most common complication and the main cause of conversion to total hip arthroplasty (THA) following the fracture of acetabulumThe pathogenesis of PTOA is still unclear. Despite cadaveric specimen studies and finite element analysis revealing some biomechanical characteristics of the acetabular fractureset al. reported an in vivo Nubian goat model for simulating human posterior wall fracture of acetabulumFor comprehensive research, a reproducible animal model with consistent fracture pattern and cartilage trauma is prerequisite but also very difficult. During the past decades, most animal models simulated intra‐articular fractures by osteotomies without impaction of cartilageThe goals of the present study were: (i) to develop a post‐traumatic osteoarthritic model of hip following fracture of acetabulum in rabbit for revealing biochemical mechanism of PTOA; (ii) to explore the macroscopic and radiographic changes of the fractured hips in different groups as time progresses; and (iii) to preliminarily assess the effect of open reduction and internal fixation surgery after dorsal wall fracture of acetabulum.Thirty‐six unselected skeletally mature male New Zealand white rabbits  were purchased from WQJX Bio‐technology Co. Ltd. . Rabbits were individually housed in single cages in a 12/12‐h light/dark cycle, fed with standard laboratory rabbit chow and tap water ad libitum. All procedures performed in the present study involving animals were conducted in accordance with the recommendations of Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Ethics Committee of the General Hospital of Central Theater Command (No: 2017019). Radiographs of the pelvis and femur were made to verify closure of the growth plate and no limb abnormality while normal gait was examined. After 2 weeks of acclimation, rabbits were equally divided into the sham group (n = 12), the non‐ORIF group (n = 12), and the ORIF group (n = 12) in accordance with the random number table.Rabbits in non‐ORIF and ORIF groups had survival surgeries under aseptic condition to create acetabular fractures of dorsal wall, simulating dashboard impaction mechanism. No more modeling procedure was made after joint exposure for the sham group Fig. . All surFor each rabbit, general anesthesia was induced through intravenous injection of pentobarbital sodium (30 mg/kg). After shaving the hair and preparing skin of the left haunch, rabbit was positioned in right lateral recumbency on an operating table, with the left hip uppermost.The left hip was exposed through a posterior approach. A 5 cm curved incision was made centered on the vertex of greater trochanter, the fascia between the superficial gluteal muscles was incised and deep muscle was split by blunt dissection and retracted to expose the retroacetabular surface. For a few cases, partial tenotomy of the short external rotators, which inserted to the medial surface of greater trochanter, should be carried out to get sufficient field as needed.A 0.8 mm Kirschner wire drill was used to outline a proposed trapezoid fracture fragment Fig. . The widThe rabbit was fixed in supine position on a customized worktop with an oblique plane of 30°. The left hip was flexed at approximately 120° and the ipsilateral knee was kept at natural flexion with a plastic brace, so that the axis of the femoral shaft was perpendicular to the center of the proposed dorsal fragment of acetabulum as well as the ground. According to Judet's research on the posterior wall fracture mechanismA customized drop‐tower impacting configuration was applied. The left knee was tightly enfolded by a customized plastic brace. The slide‐rod was kept in line with the femoral shaft as well as perpendicular to the floor. A rubber pad which fitted perfectly with the surface of the plastic brace was fixed between the end of the slide‐rod and the brace surface to ensure a uniform impaction. While an assistant kept the operated limb at right position, a one kilogram of gravity‐accelerated mass slid down from 1 meter height along the slide‐rod and impacted the left knee Fig. .After impaction, the animal was returned to the operating table and the left hip was inspected. For the ORIF group, the displaced fragment and other debris or dorsal dislocation, if existed, were reduced under direct vision and fixed with a six‐hole, 1.5 mm reconstruction plate system. What was noteworthy was that the plate must be contoured elaboratively to provide a matched buttress for the dorsal wall fragment, which would be kept at anatomic reduction and stable during postoperative daily activity. The plate was secured with two 1.5‐mm bicortical screws above and below the fragment and care was taken to ensure all of the screws were extra‐articular. Moreover, the sciatic nerve, which was no far away from caudal acetabulum, should be carefully protected during operation. The left hip was gently moved in full range of motion (ROM) and it was ensured that there were no abnormal sounds, which was correlated with intra‐articular penetration of screws. For the non‐ORIF group, the fragments were left as displaced, but transverse fracture or dislocation, if they existed, should be reduced to recover their natural position. The stability of the femoral head and the fractures were also inspected. The wound was rinsed with saline and the fascia was closed with single‐knot stitches followed by a continuous suture for skin. Postoperative radiographs were used to assess the models and quality of reduction.After recovering in a warm room, the rabbits were sent back to their individual cages and immediately allowed full weight‐bearing postoperatively. Cefazolin sodium  was administered as prophylactic antibiotics preoperatively and for the initial 3 days after surgery. Buprenorphine  was administered twice daily for 5 days as postoperative analgesia.Except for the ORIF group at 3 weeks, in which three rabbits were euthanized since one rabbit died of infection, every four animals for each other group were euthanized at 3, 6 weeks and 6 months, respectively, by overdose of pentobarbital sodium .After removing the hardware, anteroposterior digital radiographs of the pelvis were taken, then left proximal femur and acetabulum of each rabbit were harvested and surrounding soft tissue was meticulously removed. Immediately after taking photographs using a digital camera , the specimens were fixed in neutral buffered formalin.According to the photographs of harvested joints, morphological characteristics such as cartilage degeneration and remodeling of acetabulum or femoral head were recorded and preliminarily compared between different groups at different time points.Based on the features of hip joint radiographs, a semi‐quantitative scoring system , pairwise comparisons were completed using the Mann–Whitney U tests controlling for type I error across tests with the Bonferroni adjustment and P values less than 0.0167 would be considered as statistically significant.Statistical analysis was performed using the Statistical Package for the Social Science software , the semi‐quantitative scores were calculated and examined with independent‐sample Kruskal–Wallis H tests. For those analyses in which the overall test was significant , in which one was simultaneously accompanied by dorsal dislocation and another one presented an enlarged fragment that involved all of the caudal part of acetabulum. The other 21 were pure dorsal‐wall fractures as proposed  in non‐ORIF group kept limping to some extent and restricted joint movement, in particular with extension and abduction. Two weeks after operation, all of the 35 alive rabbits were examined to check the force of the traumatized limbs and compared to the contralateral ones. There was no obvious muscle weakness when they kicked, which correlated with sciatic nerve injury.Three weeks after surgery, articular capsules and glenoid labra of animal models showed mild thickening, in particular in the non‐ORIF group Fig. . In the At 6 weeks, all fractures healed and there were a lot of osteophytes around the non‐ORIF acetabula while capsules and glenoid labra further thickened. The fragments healed in displaced position and were partially absorbed. The cartilage defects were filled by what appeared to be fibrocartilage with uneven articular surface. Characteristics of cartilage degeneration were evident in the joint as well obvious abrasions located on femoral heads that were opposite to the previous fracture sites. Comparatively speaking, cartilage degeneration and structure of the joint were kept in much better condition in the ORIF group, though not as good as in the sham group.The non‐ORIF group showed hypertrophic hip joints while capsules and glenoid labra were severely thickened and stiffened at 6 months. The structures of acetabular fossae were almost impossible to be identified and there were some cartilage defects with subchondral bone appearance at the residual fossae. Besides of the yellowish cartilage, the spheric femoral heads were flattened and malformed as a result of long‐time wearing and remodeling. On the contrary, acetabula and femoral heads of the ORIF group had no significant further changes when compared to that their condition at 6 weeks.As the radiographs showed Fig. , there wAt 6 weeks, compared to the sham group, the non‐ORIF rabbits demonstrated uneven radiodensity near the previous fractured sites. Regional sclerosis was accompanied by osteophytes growing. One rabbit in this group represented mild femoral head remodeling. Other than radiolucent holes left by screws, there were limited osteophytes which were much smaller than that of non‐ORIF group at the caudal edge of acetabula in the ORIF group.The radiographs of the non‐ORIF group showed malformed acetabula with shallow fossae and large osseous bodies occupied the joint space at 6 months. Furthermore, long‐time bearing and further joint remodeling led to flattened femoral heads with coxa‐femoral subluxation. Osteophytes expanded around the joint. Subchondral bone of acetabula and femoral heads became obviously sclerotic. Compared to the radiographs of prior intervals, the subchondral bone of acetabula in the ORIF group showed mild sclerosis at 6 months and not much more growth of osteophytes.P = 0.011, <0.0167), while there was no statistical significance between the sham and ORIF groups. Although both non‐ORIF and ORIF had significantly higher scores when compared to the sham group at 6 weeks , there was no significant difference between the two modeled groups. At 6 months, one acetabulum in the sham group was given a score of 1 for minimal periarticular osteophyte that was considered as natural degeneration. At this interval, three quarters of the rabbits in the non‐ORIF group were given the most severe scores of 3 and showed statistically significant difference when compared to the sham group , but no significant difference was observed between the ORIF and sham groups or the non‐ORIF and ORIF groups.There were trends toward increasing radiographic scores according to the radiographic scoring system in the non‐ORIF and ORIF groups from 3 weeks to 6 months Fig. . Three wet al. developed an in vivo Nubian goat model for simulating human posterior wall fracture of acetabulum, like some other large animal models, this model may be limited by its high cost, long degenerative course, restriction of experiment condition, and the difficulty in carrying out large sample studiesStudies investigating potential pathogenesis and pathology of post‐traumatic osteoarthritis following fractures of acetabulum and evaluating the efficacy of therapeutic treatments requires subjects with hips which are large enough to apply surgical interventions. Small species such as mice, guinea pigs, and Syrian hamsters suffer from their limited joint sizes and operations are technically demanding or impossible. Although Olson et al. demonstrated that rabbits mimicked many characteristics of human osteoarthritis such as degenerating with aging and increased risk with weightAriz et al., a posterior wall fracture happens when the hip is positioned at approximately 0° of abduction in the frontal plane and the exact location of the main fragment varies with the amount of flexion of the hip at sagittal planeAs the most common fracture pattern of acetabulum, posterior wall fracture always happens in motor vehicle crasheset al. describedThe fracture was designed to be a trapezoid fragment in order to mimic the usual crescent‐shaped posterior wall fracture in humans as Waller et al. reported an approximate 25 MPa stress would destroy the cartilage tissueet al. revealed that a stress more than 40 MPa would result in comprehensive cartilage matrix damage and full‐thickness chondrocytes death (80%–100% of chondrocytes) in an in vivo rabbit modelBesides, drilling and connecting procedures during outlining a fragment should only pass through the outer cortex of the retroacetabular surface, so that articular cartilage was kept intact and would get sufficient support from surrounding tissues. When the gravity accelerated mass impacted to the knee, blunt trauma would happen to the cartilage surface. Repo In the present study, 21 out of 24 (87.5%) acetabula presented with proposed fractures and the other three were associated fractures  accompanied by dorsal dislocation or not, which may be correlated with individual variation such as microstructural change and bony strength. The fracture model demonstrated a high consistency and reproducibility.et al. also revealed a negative relationship between PTOA and the quality of reductionAs the macroscopic and radiographic changes showed, the hips in two modeled groups revealed progressive degeneration as the time progressed when compared to the sham group. At the end interval of the study, the hips from the non‐ORIF group exhibited severely malformed and hypertrophic joints, which was consistent with the lost function of the traumatic limbs and the trend of increased radiographic scores as the semi‐quantitative system showed. These kind of degenerative changes may be the result of different biomechanical environments with changed load distributionThere are several limitations to the present study. First, in addition to the magnitude of load, injuries would also be affected by loading rateThe fracture model with high consistency and reproducibility showed progressive post‐traumatic osteoarthritic changes, which could be improved by an open reduction and internal fixation surgery. The model provided an alternative selection for investigating potential pathogenesis and pathology of post‐traumatic osteoarthritis following fracture of acetabulum.


Lignin is a polyaromatic polymer contained in plant cell walls, and it is considered the most abundant noncarbohydrate polymer on Earth. The aromaticity and richness of its functional groups render lignin an attractive starting biomacromolecule for conversion into a variety of value‐added products. The development of successful strategies for lignin valorization infers the design of effective depolymerization protocols. Most research on lignin depolymerization has focused on batch‐mode processing, whereas only a few studies have investigated such lignin transformations in continuous reactor systems. In this Concept, emerging developments within the concept of continuous lignin processing and the challenges remaining in realizing the efficient valorization of lignin by using this technology are highlighted. A special focus is set on the hydrothermal conversion of technical lignin under continuous‐flow conditions, together with suggestions for future research and technology development. Ready for the flow: The latest advances in the field of hydrothermal depolymerization of technical lignin under continuous‐flow conditions are summarized and the challenges remaining in realizing the efficient valorization of lignin by using this technology are underlined. The production of renewable fuels and chemicals from biomass‐based resources is essential to drive the transition to a more sustainable chemical industry. Among different routes offered by nature to catalyze the transition, lignin, being the most abundantly available biopolymer, fosters numerous opportunities for producing valuable aromatic building blocks. Lignin is also a major industrial side product generated by pulp and paper mills and cellulosic ethanol production, presenting a bulk feedstock referred to as technical lignin.The aim of this Concept is to briefly highlight the latest developments in the area of continuous lignin depolymerization. The hydrothermal depolymerization of technical  lignin streams into low‐molecular‐weight aromatic compounds is particularly underlined. A performance comparison between batch and continuous systems for lignin depolymerization is presented, pointing out the major challenges encountered in both operations. A variety of emerging studies have been reported on other continuous lignin depolymerization strategies, such as hydrogenation and lignin‐first biorefining.6, 7 HIn the last decade, very few research initiatives have tackled the development of continuous‐flow reactor systems for the hydrothermal depolymerization of technical lignin into valuable phenolic compounds Figure . In the Roberts et al.3) filled with ZrO2 pellets using K2CO3 as a cocatalyst and phenol as both a cosolvent and a capping agent. The products consisted of an aqueous phase, containing phenolics, and a bio‐oil, with an increased heat value (32 MJ kg−1) in reference to the lignin feed. Otromke et al.Nguyen et al.It should be noted that the aforementioned studies were all carried out using plug‐flow reactor (PFR) modules. Recently, Kristianto et al.As regards the chemical structure, technical lignins are characterized by being very complex and highly heterogeneous. The inherent heterogeneity of native lignin is additionally coupled with the variability and the complex chemistries of the delignification processes used in its isolation, for example, kraft pulping.cis,cis‐muconic acid and 2,4‐ or 2,5‐pyridinedicarboxylic acid.Further challenges can occur because of the rather high degree of complexity, diversity, and variability of the generated product mixtures. Such products are seldom suitable for direct utilization, and innovative separation and upgrading processes are thus required. Membrane separation of depolymerized lignin represents a promising approach for product fractionation and upgrading, by which well‐defined functional fractions could be obtained.17, 18Understanding and improving technical lignin transformations under continuous‐flow conditions could facilitate the transition towards industrial application. The continuous depolymerization of lignin has a number of advantages compared to batch‐mode processing Figure . ContinuIn light of the work summarized above, the authors strongly feel that the continuous processing concept is an important stepping‐stone to technical lignin valorization. New processes with improved sustainability profiles could potentially be enabled for bio‐based chemical production on an industrial scale. Further research is needed to include optimization of the operating conditions, addressing the formation and removal of carbon deposits, reactor design for scale‐up, design of heat recovery solutions, and perhaps also process intensification solutions with the ability of cofeeding hydrogen or oxygen‐rich gas to the reactors.The authors declare no conflict of interest.


Public health organisations use public health indicators to guide health policy. Joint analysis of multiple public health indicators can provide a more comprehensive understanding of what they are intended to evaluate.To analyse variaitons in the prevalence of congenital anomaly‐related perinatal mortality attributable to termination of pregnancy for foetal anomaly (TOPFA) and prenatal diagnosis of congenital anomaly prevalence.We included 55 363 cases of congenital anomalies notified to 18 EUROCAT registers in 10 countries during 2008‐12. Incidence rate ratios (IRR) representing the risk of congenital anomaly‐related perinatal mortality according to TOPFA and prenatal diagnosis prevalence were estimated using multilevel Poisson regression with country as a random effect. Between‐country variation in congenital anomaly‐related perinatal mortality was measured using random effects and compared between the null and adjusted models to estimate the percentage of variation in congenital anomaly‐related perinatal mortality accounted for by TOPFA and prenatal diagnosis.The risk of congenital anomaly‐related perinatal mortality decreased as TOPFA and prenatal diagnosis prevalence increased . Modelling TOPFA and prenatal diagnosis together, the association between congenital anomaly‐related perinatal mortality and TOPFA prevalence became stronger . The prevalence of TOPFA and prenatal diagnosis accounted for 75.5% and 37.7% of the between‐country variation in perinatal mortality, respectively.We demonstrated an approach for analysing inter‐linked public health indicators. In this example, as TOPFA and prenatal diagnosis of congenital anomaly prevalence decreased, the risk of congenital anomaly‐related perinatal mortality increased. Much of the between‐country variation in congenital anomaly‐related perinatal mortality was accounted for by TOPFA, with a smaller proportion accounted for by prenatal diagnosis. How can we analyse inter‐linked public health indicators?Public health indicators are commonly used to inform policy decisions. Public health indicators in the same area are often linked, meaning variation in one indicator may be associated with variations in another. Nevertheless, public health indicators are usually considered separately.Using multilevel analysis, we present a novel method for modelling the association between public health indicators that takes into account the hierarchical nature of data used to measure health indicators. Specifically, the method can be used to explore how, and the extent to which, different public health indicators may be related to one another.1Public health indicators are commonly used to assess a population's health status. They are intended as an effective and uncomplicated way to compare public health between populations and over time.Congenital anomalies are an important public health concern given that they are major contributors to infant mortality, morbidity in childhood and lifelong disability.EUROCAT have developed six public health indicators that aim to evaluate the public health impact and assessment of interventions for congenital anomalies in Europe, using data routinely recorded by each registry.Outside of the perinatal setting, previous studies have examined the association between several public health indicators relating to a range of conditions.The aim of this study was to demonstrate a multilevel statistical method of analysing inter‐linked public health indicators using an interesting example of EUROCAT public health indicators. Using our proposed method, we will show how to quantify what proportion of between‐country variation in congenital anomaly‐related perinatal mortality may be accounted for by TOPFA prevalence. We will also test the hypothesis that increased TOPFA prevalence is associated with decreased congenital anomaly‐related perinatal mortality rates.22.1Data on congenital anomalies notified to 18 full member EUROCAT registers in 10 countries between 1 January 2008 and 31 December 2012 were included in this study  following prenatal diagnosis of a congenital anomaly, divided by the total number of live births and stillbirths in the country of interest, multiplied by 1000.The prevalence of termination of pregnancy for foetal anomaly (TOPFA) per 1000 total birthsThe prevalence of prenatal diagnosis of a congenital anomaly per 1000 total births: defined as the number of prenatally diagnosed cases with a congenital anomaly divided by the total number of live births and stillbirths in the country of interest, multiplied by 1000.The following indicators were used for each included country:2.3j (Model 0):λ is the congenital anomaly‐related perinatal mortality indicator that varies across countries, as represented by the index j. The variance term τ00 represents “baseline” variations in the prevalence of perinatal mortality between countries.Prevalence rates for each indicator were calculated by country, with the corresponding 95% Poisson confidence intervals (CI). A null multilevel Poisson model  was fitted with congenital anomaly‐related perinatal mortality prevalence as the outcome  and a random effect for country, represented by the index In Model 1, the univariable association between (continuous) TOPFA prevalence and congenital anomaly‐related perinatal mortality prevalence was modelled:In model 2, the univariable association between (continuous) prenatal diagnosis of congenital anomaly prevalence and congenital anomaly‐related perinatal mortality prevalence is modelled:It is also possible to fit a multivariable model, in order to control for additional variables. To demonstrate, Model 3 was fitted with both our explanatory variables included:γ01 and γ02 from the fixed effects part of the model can be interpreted as incidence rate ratios (IRRs), for example γ01 in model 1 can be interpreted as the relative risk of congenital anomaly‐related perinatal mortality per unit increase in TOPFA prevalence. These show how the outcome and explanatory indicators are associated, for example the IRRs in models 1 and 2 can test the hypothesis that the risk of congenital anomaly‐related perinatal mortality decreases as TOPFA and prenatal diagnosis of congenital anomaly prevalence increase. In summary, the fixed effects can be used to test hypotheses about the association (linear or otherwise) between the outcome indicator and the explanatory indicators.We can now use the models to give us two different types of information. Firstly, τ00∗ψ) from the baseline between‐country variance (τ00). The difference between the baseline and residual variance is then divided by the baseline variance in order to calculate a percentage of the total variance:Secondly, the degree of variation in the outcome indicator that is “explained” by the explanatory indicator(s) can be calculated from the random effects part of the model. The variation between countries in the null model, which we refer to as the “baseline” variation, can be compared with the residual variation that remains after adjusting for explanatory indicators. This value can then be expressed as a percentage of the “baseline” variation between countries. So in our case, we can subtract the between‐country variation after taking into account TOPFA and prenatal diagnosis of congenital anomaly prevalence .2.4Prenatal diagnosis data were not available for Antwerp at the time of analysis, and therefore, the prenatal diagnosis rate for Hainaut was used for Belgium. Prenatal diagnosis was not available for Dublin, and therefore, prenatal diagnosis prevalence for Ireland relates to Cork and Kerry and SE Ireland only.2.5Data were downloaded in aggregate form, and therefore ethical approval was not required for this study. However, each EUROCAT register has their own ethical approvals in place for the collection of data without consent.3The prevalence of perinatal mortality in cases of congenital anomalies ranged from 0.5  per 1000 total births in Spain to 3.1  per 1000 total births in Malta   of 0.28 . In the model including only TOPFA (Model 1), the between‐country variation (τ00∗) was 0.07 , meaning the prevalence of TOPFA accounted for 75.5% of the variation in congenital anomaly‐related perinatal mortality between countries (Table (τ00ψ) was 0.17 , meaning the prevalence of prenatal diagnosis accounted for 37.7% of the between‐country variation in perinatal mortality. In the model adjusted for TOPFA and prenatal diagnosis of congenital anomaly prevalence (Model 3), the between‐country variation (τ00∗ψ) was 0.05 , meaning 83.0% of the variation in congenital anomaly‐related perinatal mortality was accounted for.The prevalence of congenital anomaly‐related perinatal mortality in the null model (Model 0) varied between countries with a “baseline” between‐country variance .Additionally, we pooled the indicators over 4 years meaning changes in the prevalence of congenital anomaly‐related perinatal mortality, prenatal diagnosis of congenital anomaly and TOPFA were averaged over time. Although we studied a relatively short study period (2008‐12), this may have introduced some confounding of trends over time. With advances in prenatal diagnosis and medical and surgical interventions for congenital anomalies, congenital anomaly‐related perinatal mortality may have decreased and the associations with TOPFA and prenatal diagnosis may have altered in more recent years. Prevalence rates for the EUROCAT public health indicators discussed in this paper are updated annually on the EUROCAT website  and infant deaths among cases of congenital anomaly after examining graphically their two indicators of interest.5We have demonstrated a multilevel approach for quantifying the degree of variation in one public health indicator that may be attributable to another. In our example, we found a negative association between TOPFA rates and perinatal mortality among cases of congenital anomaly, which accounted for 75.5% of between‐country variation. Prenatal diagnosis with TOPFA and prenatal diagnosis modelled together accounted for 83% of between‐country variation in perinatal mortality.None.


Rangifer genealogies spanning from 15,000 years ago to the 18th century, as well as modern samples from the wild Taĭmyr population and from domestic herds managed by Nenetses. The wild and the domestic population are the largest populations of their kind in Northern Eurasia, and some Nenetses hold their domestic reindeer beside their wild cousins. Our analyses of 197 modern and 223 ancient mitochondrial DNA sequences revealed two genetic clusters, which are interpreted as representing the gene pools of contemporary domestic and past wild reindeer. Among a total of 137 different mitochondrial haplotypes identified in both the modern and archaeological samples, only 21 were detected in the modern domestic gene pool, while 11 of these were absent from the wild gene pool. The significant temporal genetic shift that we associate with the pastoral transition suggests that the emergence and spread of reindeer pastoralism in Northwestern Siberia originated with the translocation and subsequent selective breeding of a special type of animal from outside the region. The distinct and persistent domestic characteristics of the haplotype structure since the 18th century suggests little genetic exchange since then. The absence of the typical domestic clade in modern nearby wild populations suggests that the contemporary Nenets domestic breed feature an ancestry from outside its present main distribution, possibly from further South.Just as the domestication of livestock is often cited as a key element in the Neolithic transition to settled, the emergence of large‐scaled reindeer husbandry was a fundamental social transformation for the indigenous peoples of Arctic Eurasia. To better understand the history of reindeer domestication, and the genetic processes associated with the pastoral transition in the Eurasian Arctic, we analyzed archaeological and contemporary reindeer samples from Northwestern Siberia. The material represents  This manuscript demonstrates that the reindeer mitochondrial genome in Northwestern Siberia has gone through massive genetic replacement since medieval times characterized by a significant loss of native mtDNA haplotypes, together with a significant introduction of new haplotypes. The significant temporal genetic shift associated with the pastoral transition suggests that the emergence and spread of reindeer pastoralism in Northwestern Siberia involved the translocation and breeding of a special type of animal with a partly non‐native origin. Just as the domestication of sheep, goat, cattle, and pigs were critical elements in the Neolithic transition in many parts of Western Eurasia  are distributed throughout most of the northern Holarctic region with the domestic form mainly confined to the Eurasian continent. Among the world's total stock of nearly 2,000,000 domestic reindeer, ~2/3 are distributed in Russia and 1/3 in Fennoscandia —have been dated to the same period .The domestic reindeer studied are from nomadic herds managed by indigenous Nenetses Figure . NenetseKrupnik, . BeginniKrupnik, . Today NII and Ib) and Northwestern Russia , the Nenets Autonomous District (NAD), and the eastern portion of the Komi Republic (KR), with ages spanning from ~15,000 years before present (BP) to ~300 BP  is from reindeer close to Malygina Strait at the north end of the I͡Amal Peninsula. Their seasonal migration pattern is mainly within the northern territory of I͡Amal sovkhoz. The Taz‐Nenets samples (sample code 18) were from a herd in the Taz District located directly east of the I͡ANAD on the opposite side of the Ob' Gulf from the I͡Amal Peninsula. These eastern Nenets families move their herds annually from the town of Tazovsk on the Taz River all the way to the western part of the TDND. The Eniseĭ‐Nenets samples (sample code 19) were from the reindeer herding families who travel around the settlements of Tukhard and Nosok located at the western part of TDND. Most of the ~100,000 Nenets reindeer in TDND are situated in this area.DNA from 173 skin, muscle, and blood samples from contemporary domestic and wild reindeer were extracted and analyzed. The four domestic sample sets were from separate and widely spread districts in I͡ANAD and the western part of the Taĭmyr Dolgano‐Nenets District  following the protocol described in Bjørnstad and Røed  following the manufacturer's protocol. A 503‐bp long fragment of mitochondrial CR was amplified and sequenced as given in Kvie, Heggenes, and Røed . All seq2.4arlequin v3.5  of sample sets, that is, codes 1–14 together with 20 versus codes 15–19 on the basis of the among‐group partitioning of molecular variance , nucleotide diversity  and nucleotide pairwise differences  in the recent domestic sample set, compared to the wild sample set  and in Northwestern Russia  and with all other haplotypes radiating from this by one to three mutations  or in the older sample codes 3–13 (n = 37). Subcluster Ib contained three haplotypes in 11 samples, two in modern domestic reindeer and nine in ancient reindeer (sample codes 3–12).The Bayesian phylogenetic tree reconstruction of the 137 haplotypes identified several medium to well‐supported clusters/subclusters Figure . Some ofs Figure . Such a s Figure . This an4Our analysis of the mitochondrial CR in contemporary and archaeological reindeer in the vast Nenets‐occupied region reveals two distinct genetic clusters. The first corresponds to the modern wild reindeer together with the older sample sets ranging from the late Pleistocene to the late medieval periods covering large areas from I͡Amal in the East to the Ural Mountains in the West. The second includes those from widely separated contemporary domestic herds from I͡ANAD and the western part of the TDND together with the relatively recent archaeological site dated to 300 BP. The high proportion of temporal sample overlaps on I͡Amal and the fact that nearly all archaeological sites clustered together with contemporary wild reindeer, and all modern domestic herds assigned to the other cluster, gives strong evidence that these highly differentiated clusters represent the maternal gene pools of wild and modern domestic reindeer respectively. Based on these results, the mitochondrial CR appears to be an appropriate marker to study genetic processes associated to the pastoral transition in Northwestern Siberia.4.1The high levels of genetic variation within a homogeneous genetic structure found within the wild gene pool both temporally and spatially suggest that relatively large populations of wild reindeer occupied the region since late Pleistocene. The significant genetic shift associated with the late‐dated archaeological site Khali͡ato‐1 is striking and points toward a fundamental genetic change on the I͡Amal that seems to have been completed by the start of the 18th century. This change coincides very roughly with the period associated with the onset of the pastoral tradition. Using historical sources, Krupnik  associatIt should be noted that others have interpreted the many artifacts found at sites on the I͡Amal Peninsula as signs of a social complexity and a remarkable inter‐regional standardization of techniques built on large‐scale reindeer transport and large‐scale reindeer husbandry, beginning as early as 2,000 BP  at sites 3, 5, 10 and 13 could indicate that a small number of animals with a genetic constitution similar to those of the domestic reindeer today were present on I͡Amal since as far back as 6,000 BP. If local wild reindeer were tamed , and the small fraction haplotypes belonging to the domestic lineages among the archaeologic sites 1–14, nevertheless suggest that the dominant domestic type of today had its origins in animals imported from other areas.Several mechanisms could have been involved in introducing new maternal genomes to the region. This polar region experiences periodic crashes in the number of reindeer due to poor weather or spring icing events ; data curation (lead); formal analysis (lead); funding acquisition (lead); investigation (lead); methodology (lead); project administration (lead); resources (supporting); software (supporting); supervision (supporting); writing‐original draft (lead); writing – review and editing (lead). Kjersti Kvie: Data curation (supporting); formal analysis (supporting); resources (supporting); writing‐original draft (supporting); writing – review and editing (supporting). Robert J Losey: Resources (supporting); writing‐original draft (supporting); writing – review and editing (supporting). Pavel A Kositsev: Resources ; validation (supporting); writing‐original draft (supporting); writing – review and editing (supporting). Anne K Hufthammer: Data curation (supporting); resources (supporting); validation (supporting); writing‐original draft (supporting); writing – review and editing (supporting). Mark J Dwyer: Resources (supporting); writing‐original draft (supporting). Vasiliy Goncharov: Resources (supporting); visualization (supporting); writing‐original draft (supporting). Konstantin B Klokov: Resources (supporting); writing‐original draft (supporting). Dimitry V Arzyutov: Resources (supporting); Writing‐Original Draft (Supporting); Writing‐Review & Editing (supporting). Andrei Plekhanov: Resources (supporting); writing‐original draft (supporting). David George Anderson: Funding acquisition (supporting); project administration (supporting); resources (supporting); writing‐original draft (supporting); writing – review and editing .Appendix S1Click here for additional data file.


MDS‐criteria for clinical diagnosis of progressive supranuclear palsy (PSP) were recently published, their usability in a classical clinical setting is yet unknown. We retrospectively applied the new criteria using PSP patients’ case files. Assignment of PSP diagnosis according to the MDS‐criteria was possible in 57/80 cases. The main difference to former specialist classification was a lower phenotype diversity and higher representation of PSP‐RS. Furthermore, we examined those patients’ brain MRIs. While neuroradiologists’ reports were suggestive of PSP only in 11/62, the analysis of a blinded rater revealed pathological midbrain‐to‐pons‐ratio in 40/62 implying this imaging feature is often missed. The Movement Disorders Society (MDS) Criteria for the clinical diagnosis of progressive supranuclear palsy were recently published.The new MDS criteria aim on “improving the clinical detection of underlying PSP pathology by maintaining high diagnostic sensitivity for PSP‐RS, improving sensitivity for early and variant PSP presentations, and achieving high specificity versus alternative diagnoses”.Only few data exist yet on the usability and practicability of the new MDS criteria in a classical movement disorder clinical setting.We sought to determine i) whether the new criteria including the MAX rules are suited to retrospectively assign PSP patients to the respective clinical predominance types and ii) whether and how the new classification differs compared to a movement disorders specialist diagnosis and to the NINDS‐SPSP criteria, respectively.We performed a single‐center retrospective analysis of case files of movement disorders specialist care. The study was approved by the local ethics committee of the Technische Universität Dresden EK393082019). We first screened for patients with the clinical diagnosis of PSP who were admitted to our movement disorders center at the Department of Neurology of the University Hospital Dresden from 2006 to 2017. Case files were then reviewed for the following items: Age, sex, admission diagnosis, in‐house movement disorders specialist diagnosis and PSP phenotype,9. We firWe assessed both brain MRI reports and scans (complete image datasets available in 62/80 patients) for findings suggestive of PSP. A blinded rater (K.P.) retrospectively analyzed those brain scans using the midbrain‐to‐pons ratio in the midsagittal plane of conventional MRI.Data are shown as mean ± standard deviation (SD).We identified 80 patients with the inhouse movement disorders specialist diagnosis of PSP who were admitted to our movement disorders unit from 2006 to 2017 . 55/80 patients with the movement disorder specialist diagnosis of PSP were misdiagnosed before admission, whereas Parkinson’s disease was the most frequent suspected diagnosis Fig. . The meaIt was possible to assign PSP diagnosis and predominance type according to the new MDS criteria in 57/80 compared to 29/80 cases according to NINDS criteria. Lack of information about postural stability and onset of falls was the main reason for failed retrospective diagnosis and subgroup allocation in both, the MDS and the NINDS criteria. The strict time criteria for the onset of falls within the first year of disease was the main reason for the higher rate of missed retrospective diagnosis of PSP using NINDS criteria. All 29/80 patients diagnosed according to NINDS criteria were classified as PSP‐RS using the MDS criteria.Prior to applying MAX rules, we encountered difficulties when multiple symptoms in one or even more functional domains were present in one individual patient . This was independently reproduced by Ali and colleagues.Finally, we investigated the frequency of midbrain atrophy as a typical MRI sign for PSP.The MDS‐PSP criteria work well for retrospective allocation to PSP subgroups and were more suitable for identification of PSP in comparison to the former NINDS‐SPSP criteria mainly due to the wider time span of 3 years for the clinical domain of postural instability and falls. However, this also results in overemphasizing postural instability as a rather nonspecific symptom and classifying more cases as PSP‐RS . JL has received honoria for presentations/advisory boards from Bayer Healthcare and mediaire. Andreas Hermann has received funding from the Innovationsfond des Gemeinsamen Bundesausschusses, the Helmholtz‐Association and Hermann und Lilly Schilling‐Stiftung für medizinische Forschung im Stifterverband. He has received honoraria for presentations/advisory boards from Desitin and Biogen. He has received royalties from Elsevier Press. He serves as an editorial board member of BMC neurology.The study was approved by the institutional review board at the Technische Universität Dresden (EK393082019) and in accordance with the Declaration of Helsinki.Table S1. Application of the MDS‐PSP criteria in our single‐center cohort showing symptoms, movement disorders specialist diagnosis and possible diagnoses before application of the multiple allocation extinction rules (MAX) as well as the midbrain to pons ratio and MRPI.Click here for additional data file.


The recent development of the CRISPR/Cas9 system as an efficient and accessible programmable genome‐editing tool has revolutionized basic science research. CRISPR/Cas9 system‐based technologies have armed researchers with new powerful tools to unveil the impact of genetics on disease development by enabling the creation of precise cellular and animal models of human diseases. The therapeutic potential of these technologies is tremendous, particularly in gene therapy, in which a patient‐specific mutation is genetically corrected in order to treat human diseases that are untreatable with conventional therapies. However, the translation of CRISPR/Cas9 into the clinics will be challenging, since we still need to improve the efficiency, specificity and delivery of this technology. In this review, we focus on several in vitro, in vivo and ex vivo applications of the CRISPR/Cas9 system in human disease‐focused research, explore the potential of this technology in translational medicine and discuss some of the major challenges for its future use in patients. It is revolutionizing the biomedical research field and holds promise to treat or prevent many human genetic disorders. The perfect genome‐editing tool has to be able to alter a genomic sequence efficiently, showing high DNA sequence specificity, and minimal off‐target effects.Nevertheless, this approach has several limitations, such as its low editing efficiency and unwanted genome‐editing events where the donor DNA template is more frequently inserted into the genome randomly than at the desired location.Alternatively, site‐directed zinc finger nucleases (ZFs)The development of the CRISPR/Cas9 system has proven to be a major scientific breakthrough and made gene editing more accessible. Distinct from the protein‐guided DNA cleavage used by TALENs and ZFs, CRISPR/Cas9 depends on a small RNA to introduce a site‐specific DSB.The simplicity of CRISPR/Cas9 technology coupled with a unique DNA cleaving mechanism, the ability to target multiple regions, and the existence of different type II CRISPR‐Cas system variants, has enabled notable progresses using this cost‐effective and user‐friendly technology to precise and efficiently modify the genomic DNA of a wide collection of cells and organisms.2Escherichia coli, the presence of a cluster of repetitive DNA sequences separated by variable spacer regions. Later, Mojica et al identified identical type of repeated sequences in numerous bacteria and archaea and named them Clustered Regularly Interspaced Palindromic Repeats or CRISPR.S thermophilus strain and two bacteriophages, showed experimentally that CRISPR confers adaptive immunity. In 2008, CRISPR RNAs (crRNAs) were shown to serve as guides in a complex with Cas proteins to promote phage resistance.In 1987, Ishino et al.CRISPR mechanisms are very diverse but can be mainly classified into two distinct classes, class 1 and class 2, depending on the organization of the effector protein complex. Class 1 comprehend three different types I, III and IV that are further subdivided into 15 subtypes. Distinct from class 1, that is characterized by the presence of a multi‐protein effector complex, class 2 is defined by a single‐protein effector module. This class is divided into types II, V and VI.The natural occurring type II CRISPR mechanism is a simple three‐component system  that showed promising potential to be adapted for genome editing. A major milestone came in 2012 when Doudna and Charpentier laboratories developed a simplified two‐component CRISPR/Cas9 system by combining tracRNA and crRNA into a single guide RNA (sgRNA).33.13.1.1In January 2013, three independent studies have shown that CRISPR/Cas9 mechanism could be repurposed to generate DSBs in DNA. By tweaking this naturally occurring mechanism, researchers were able to perform mammalian genome editing using DNA repair systems, including the NHEJ and the less‐frequent template HDR.3.1.2The cancer genetics field is one of the research areas in which CRISPR is having a significant impact. With CRISPR, it is now possible to quickly induce loss‐of‐function (LOF) and gain‐of‐function (GOF) mutations in tumour suppressor genes, oncogenes or other relevant players of the malignant transformation process.3.1.3Since the discovery by Yamanaka and colleagues that somatic cells could be reprogrammed into a pluripotent state, human induced pluripotent stem cells (iPSCs) have held great promise in several disease models, regenerative medicine, drug discovery and development.There are different approaches to generate isogenic disease models in iPSCs using the CRISPR/Cas9 system. For example, it is possible to generate Cas9‐mediated iPSC knockout cell lines via NHEJ that could be used to determine whether a given human mutation is indeed directly responsible for causing the disease or to simply study gene function.One of the most exciting CRISPR/Cas9 applications with relevance to human health is gene therapy, in which a patient‐specific mutation or mutations are genetically manipulated in order to provide a definitive cure.3.1.4Sall1 as a gene that contributes to reprogramming mouse embryonic fibroblasts into induced pluripotent stem cells.Besides enabling the edition of mammalian genomes, researchers have now the possibility of regulating gene expression and altering epigenetic states. Alterations to the CRISPR/Cas9 system facilitated this new feature without introducing DSBs,Conversely, dCas9 has also been utilized in genome‐wide experiments for targeted gene transcriptional repression.Overall, these dCas9 versions that allow for the activation (CRISPRa) or repression (CRISPRi) of target genes are powerful tools that can be used for functional genomic studies under different physiological and developmental conditions without creating DSBs. More recently, an in vivo study on a type I diabetes mouse model repurposed Cas9 to epigenetically induce gene activation and observed a significant improvement on disease phenotypes such as acute kidney injury and muscular dystrophy.3.23.2.1CRISPR/Cas9 technology brought a lot of excitement within the scientific community since it revolutionized how fast researchers are able to make a genetically modified animal models.3.2.2Dmd gene mutation. This experiment resulted in genetically mosaic progeny ranging 2%–100% gene correction and varying degrees of muscle phenotypic rescue.CRISPR/Cas9 gene‐editing technology has dramatically changed the way we can model and treat human disease in vivo. In order to test the capability of the CRISPR/Cas9 system to correct disease‐causing mutations, in vivo genetically modified animals can be used. In 2013, Wu et al. reported that a mouse model with a dominant cataract‐causing mutation in the Crygc gene could be corrected by co‐injecting Cas9 and a sgRNA targeting the mutant allele into zygotes. CRISPR/Cas9 system was able to correct the mutant allele via HDR pathway using an exogenous oligonucleotide or the endogenous WT allele as template and showing very little off‐target events.In 2014, another study demonstrated that using the mouse model of hereditary tyrosinemia type 1 (HT1), CRISPR/Cas9 system could be used to successfully correct a mutation in post‐natal animals.Since then, other studies have shown that CRISPR/Cas9 gene‐editing technology could be used for the in vivo treatment of other genetic disorders in adult mice. For example, three independent groups have used adeno‐associated virus (AAV) to deliver CRISPR/Cas9 machinery to the mdx mouse model of Duchenne muscular dystrophy with the goal of restoring dystrophin expression in skeletal and cardiac muscle cells.Collectively, these in vivo studies set groundbreaking work in the development of new therapies for genetic diseases. For the most part, human genetic disorders are uncurable and this is reflected on patient's poor life quality and shortened life expectancy. The significant progress made in the CRISPR/Cas9 technology enables the development of promising treatments and highlights the potential of this gene therapy approach to cure human genetic diseases.3.2.3Cardiovascular diseases (CVDs) are the number one cause of death worldwide according to the World Health Organization. The advances in CRISPR/Cas9 technology have shown to greatly impact the development of new in vivo tools that allow a better understanding of mechanisms underlying CVDs. More importantly, the expansion of CRISPR/Cas9 techniques accelerated the development of novel therapies capable of treating CVDs.Ttr) gene in the mouse liver with a single administration. The authors combined the nanoparticle system with CRISPR/Cas9 components to target the Ttr gene and observed a significant reduction in serum protein levels that persisted for at least 12 months. It will be interesting to test whether this approach will be effective and durable in other disease models other than cardiac amyloidosis.The availability of viable and efficient delivery methods represents one of the biggest challenges of translating the CRISPR/Cas9 into the clinic, as it will be discussed in more detail in the following section. Finn et al. 3.2.4The success of ex vivo gene therapy relies on the establishment of optimized protocols for culturing patient‐derived primary cells that after genome editing can be transplanted back into the patient. The hematopoietic system is an excellent target for this approach, because target cells can be easily withdrawn from the patient peripheral blood and can be re‐injected after editing and expansion.The precise selection of genetically modified cells harbouring the correct edited allele without undesirable off‐target mutations represents one of the most important aspects of ex vivo gene therapy. As the process of selection is very efficient, and only selected cells will be transferred back into the patient, the accuracy of CRISPR/Cas9 is less critical in ex vivo than in in vivo gene therapy.3.2.5CRISPR/Cas9 system is undoubtedly the most revolutionary technology in medicine over the past decades. Since researchers were able to demonstrate that CRISPR/Cas9 could be repurposed as an editing tool, this technology has developed with enormous scientific, medical and industry impact. In this review, we have covered several examples of how CRISPR/Cas9 technology had direct or indirect impact in the clinic. Despite recent and important advances, several issues have to be addressed in order to bring CRISPR/Cas9 genome‐editing tools into the clinic. One of the major obstacles for the translation of CRISPR/Cas9 into clinically useful tools is the possible off‐target effects. Several studies using a variety of approaches, including computational predictions, in vitro and in vivo high‐throughput profiling and whole‐genome sequencing methods, have reported significant rates of off‐target effects associated with CRISPR/Cas9. It is important to note that unwanted effects of CRISPR/Cas9 such as off‐target editing and off‐target binding might result in malignant transformation and other unforeseeable consequences.The development of methods that minimize off‐target effects of CRISPR/Cas9 approaches has been a major focus of research. One of these strategies requires that two separate Cas9 binding events simultaneously occur at the same locus in order to execute cleavage of DNA. The inactivation of either of the two catalytic residues within Cas9 converts the enzyme into a nickase (Cas9n) which cleaves or ‘nicks’ a single DNA strand instead of a double strand.Another major obstacle for the clinical translation of CRISPR/Cas9 is the limited efficiency of HDR‐mediated gene correction. Factors known to determine this efficiency include cell type, cell state and competition with the NHEJ. As many treatments of human genetic diseases are based on HDR‐mediated gene correction, in which a template sequence is delivered to replace the mutated version, major progress in the efficiency of HDR is necessary. Several efforts have been made to increase HDR efficacy, including the rational design of single‐stranded DNA donors.Besides its low efficiency, HDR has been considered to be mainly limited to applications in dividing cells.Streptococcus pyogenes Cas9 (spCas9) gene(4.2 Kb), the sgRNA, the donor template as well as associated promoters and regulatory sequences into AAV.Staphylococcus aureus (saCas9) allowing for the integration of a Cas9 together with a sgRNA into a single AAV.Virtually, all macromolecular therapies have to solve issues of delivery that often limit their efficacy.Pten, a tumour suppressor gene involved in NASH and a repressor of the PI3/AKT pathway. Surprisingly, this study describes the production of Cas9‐specific antibodies and the secretion of IL‐2 from splenocytes that had been engineered with Cas9 system targeting the Pten locus.Host immune responses induced by the delivery of bacterial Cas9 proteins or gene therapy vectors represent another challenge for the translation of CRISPR/Cas9 approaches into the clinic. More recently, a mouse model for non‐alcoholic steatohepatitis (NASH), a frequent liver disease in humans characterized by excessive fat build‐up in the liver, was generated using spCas9 to delete 3.3FBN1 gene involved in the Marfan syndrome, a rare autosomal dominant disorder in heterozygous embryos.The burst of CRISPR/Cas9 applications also highlighted the potential of this system and the ethical concerns associated with the possible creation of permanent and inheritable changes in the human genome. Having in mind the ethical implications, legal action was taken to delay germline genome editing. The first report using CRISPR in human embryos was back in 2015 where Liang et al. 4The CRISPR/Cas9 RNA‐guided DNA endonuclease system is a versatile technology that has rapidly transformed genome editing and basic science research. The development of improved CRISPR/Cas9 tools with high degree of DNA specificity, increased selectivity and low level of by‐products made this technology accessible to researchers worldwide to study human diseases. For example, it is now feasible to generate in vivo animal models of specific diseases in a few weeks. It is now possible to envision the treatment of genetic diseases in the near future using this technology. In fact, several clinical trials using CRISPR/Cas9 approach to treat human genetic diseases are underway (NCT03872479 or NCT03399448). However, we still need to improve the efficiency, specificity and delivery of this technology for its broader application in the clinics. A major concern that accompanies the use of CRISPR/Cas9 in the clinical setting relates to the potential risk of misusing this technology. The development of ethical and regulatory guidelines is critical to ensure that the benefits outweigh and minimize the risks. The discovery of CRISPR/Cas9 technology and its application in the clinics is a true example of the importance of bridging basic research and translational medicine. Once the CRISPR/Cas9 mechanism was unveiled, the possibilities of medical exploitation were enormous and will definitely change the way we will treat genetic disorders in the future.The authors declare that they have no conflict of interest.FV.J performed research and wrote the manuscript. WL wrote the manuscript. BI.F conceptualized the study, edited, wrote and submitted the manuscript.


The evolutionary dynamics of tumor‐associated neoantigens carry information about drug sensitivity and resistance to the immune checkpoint blockade (ICB). However, the spectrum of somatic mutations is highly heterogeneous among patients, making it difficult to track neoantigens by circulating tumor DNA (ctDNA) sequencing using “one size fits all” commercial gene panels. Thus, individually customized panels (ICPs) are needed to track neoantigen evolution comprehensively during ICB treatment. Dominant neoantigens are predicted from whole exome sequencing data for treatment‐naïve tumor tissues. Panels targeting predicted neoantigens are used for personalized ctDNA sequencing. Analyzing ten patients with non‐small cell lung cancer, ICPs are effective for tracking most predicted dominant neoantigens (80–100%) in serial peripheral blood samples, and to detect substantially more genes (18–30) than the capacity of current commercial gene panels. A more than 50% decrease in ctDNA concentration after eight weeks of ICB administration is associated with favorable progression‐free survival. Furthermore, at the individual level, the magnitude of the early ctDNA response is correlated with the subsequent change in tumor burden. The application of ICP‐based ctDNA sequencing is expected to improve the understanding of ICB‐driven tumor evolution and to provide personalized management strategies that optimize the clinical benefits of immunotherapies. Inter‐individual heterogeneity of mutation profiling is the primary obstacle in effective in vivo monitoring of mutation‐directed T‐cell killing using ctDNA sequencing. Herein, a customized sequencing strategy is developed that mainly targets predicted neoantigen‐coding mutations. This approach broadens the ctDNA sequencing coverage and is expected to improve personalized management strategies that optimize the clinical benefits from checkpoint immunotherapies. Effective and accurate monitoring of changes in tumor burden is vital for predicting responsiveness, making treatment decisions and understanding the dynamic evolution of tumor neoantigen profiles that occur during therapy.The capacity of immune checkpoint blockade (ICB) to augment tumor‐specific T‐cell cytotoxicity has resulted in impressive improvements in clinical outcomes for patients with late‐stage solid tumors.1 Desp In a longitudinal assessment, the baseline concentration and early mutational dynamics of ctDNA were correlated with radiological and survival outcomes in response to ICB treatment in multiple solid tumor types. Typically, commercially available panels for ctDNA mutation profiling cover a limited set of genes, such as known oncogenes, tumor suppressor genes, immune‐related genes, and other targets of actionability and/or research interest. However, there is mounting evidence that mutated neoantigens are the main targets of immune attack during ICB immunotherapy. Therefore, dynamic tracking of ctDNA mutations resulting in neoantigens would be of higher value than those of irrelevance. Given that T‐cell‐recognizable neoantigens are largely tumor‐specific owing to the highly patient‐dependent genetic background, personal HLA genotype, history of environmental exposure, and other factors, a predefined generic panel would face challenges in sufficiently cover these dynamics. While panel per se one might consider a “totality” approach such as whole exome sequencing (WES) or even whole genome sequencing (WGS), in reality, deep sequencing (which is required for effective mutation detection from ctDNA) of WES and WGS would become cost prohibitive. Additionally, requirement on input DNA quantity for WES or WGS makes their clinical application to ctDNA hardly practicable. Understandably, this challenge could be addressed by an individually customized panel (ICP) with a reasonable DNA input requirement and at a manageable cost, that at the same time ensures a tailored coverage of patient‐specific dominant neoantigens, with the dominant neoantigens in silico‐predicted from a one‐time sequencing of matched tumor tissue using WES.The level of circulating tumor DNA (ctDNA), a surrogate of tumor burden, can be used to estimate clinical responses in patients receiving anti‐tumor treatment.7, 8With this understanding, in this study, we designed primary tumor‐guided personalized neoantigen panels for ctDNA sequencing to monitor the clinical response to first‐line ICB treatment for patients with advanced non‐small cell lung cancer (NSCLC). We demonstrated that this strategy enables the monitoring of early response, prediction of the treatment efficacy and disease relapse, and tracking of neoantigen evolution during the course of ICB treatment.22.1FigureIt is a general perception that the spectrum of somatic mutations in tumors might vary considerably among individuals. Part of our study aimed to gain a more quantified understanding of such variability. We first investigated how many mutated genes from two patients overlapped with each other. We measured the overlap as the ratio in percentage of the number of commonly mutated genes between two patients over the total number of mutated genes across both patients. Based on 1059 patients with NSCLC in The Cancer Genome Atlas (TCGA) dataset,  A median of 28.5 (ranging from 2 to 189) dominant neoantigens was identified per patient . The top 20–30 neoantigen‐coding genes with the highest affinity scores were included into the ICP panels following to the detailed procedure in Figure S2, Supporting Information. This ICP strategy accounted for a median of 82.67% of all predicted neoantigens (ranging from 15.87% to 100%) per patient. We then performed on‐treatment monitoring of the individual based on ctDNA sequencing using their corresponding ICP. The concordance between the ICP‐detected neoantigens in ctDNA and the WES‐detected ones in tumor tissue is summarized in grid heatmaps . The numbers of overlapping genes between each commercial panel and the WES‐derived mutation spectrum for patients in our cohort were calculated. The number of overlapping genes was significantly lower for all five commercially available panels than for the ICP  or their mean values  for each patient. Furthermore, ctDNA levels showed a greater decline in patients who experienced an objective response to ICB at eight weeks after the first infusion than in patients who showed no response . We next investigated whether the early decrease in peripheral ctDNA could predict prolonged survival in response to immunotherapy, as reported in ICB‐treated cohorts with relatively large cohort sizes. Surprisingly, even with a small sample size of nine patients (P10 was excluded) with baseline blood samples in our cohort, the substantial early decrease in ctDNA abundance (≥50% decrease at eight weeks) predicted prolonged progression‐free survival after ICB administration . These observations strongly support the use of ICP‐based ctDNA sequencing to identify patients likely to have favorable outcomes following ICB treatment.Numerous studies have demonstrated that baseline ctDNA can be used as an approximate indicator of tumor burden in patients with metastases for a wide range of tumor types.22 In 2.3FigureR = 0.509–0.999, strongly supporting the potential use of ICP to monitor the dynamics of tumor burden through ctDNA sequencing . Outliers including patient 02 and patient 10 are further discussed in the following section.In addition to its use as a predictive indicator at the population level, we investigated whether the ICP‐based assessment of neoantigen level changes could be indicative of quantitative changes in tumor burden at the individual level. The radiological tumor burden response (determined by the sum of products of perpendicular diameters (SPDs)) and longitudinal ctDNA kinetics was evaluated, with the results shown in The magnitude of ctDNA decline varied substantially among individuals when measured by ICP Figure . Similar2.4FigurePatient 02 was a 68‐year‐old female diagnosed with lung adenocarcinoma and T2N1M1c stage IV disease. She was enrolled in the NEPTUNE trial (NCT02542293) that tested the efficacy of tremelimumab/durvalumab combinational immunotherapy and achieved a partial response after four cycles of administration. Baseline emission computed tomography revealed an unmeasurable metastatic lesion in the 12th thoracic vertebra 24 ands Figure .We further applied the ICP‐based ctDNA sequencing for real‐time on‐treatment biomarker monitoring and achieved improved tracking of neoantigen evolution with the following evidence: 1) Early changes in ctDNA predicted tumor load dynamics during treatment Figure . In part Immunotherapies can result in evolution of the neoantigenic landscape, consistent with the theory of immunoediting. An analysis of matched pre‐ and post‐treatment tumor samples by Valsamo and colleagues found that 7–18 putative neoantigens are eliminated in immunotherapy‐resistant cases, providing the first evidence that neoantigen evolution might be involved in acquired resistance to immunotherapy. Interestingly, in our cohort, except for that observed for one patient (Patient 10), initial neoantigen‐coding mutations could still be detected at the stage of relapse in nine patients, suggesting that a sufficiently large‐scale of neoantigen loss might be attributable to just a small portion of relapsed patients after ICB treatment. The difference in observations between Valsamo's and our study could be due to the spatial heterogenous mutation spectrum of tumors. In three of four patients in Valsamo's cohort, mutations were estimated based on matched tumor tissues sampled from proximate but different anatomical sites, and this discrepancy could be overcome by circulating assays such as ctDNA sequencing. Therefore, for patients without neoantigen loss, we propose that strategies that support or re‐provoke the activation and expansion of neoantigen‐specific T‐cells might help to overcome resistance after ICB treatment. Besides checkpoint blockade immunotherapies, neoantigen‐based personal vaccination has long been envisioned as an effective stimulus to provoke tumor‐specific T cell responses. However, despite being facilitated by the progress of high‐throughput sequencing and bioinformatic tools, the clinical effectiveness of extensively past work was far from ideal. We believe that personalized ctDNA sequencing is an excellent tool to monitor the clearance of neoantigen‐expressing tumor cells and to gain mechanistic insight into resistance to neoantigen‐based cancer vaccines.In addition to these prospects, this neoantigen‐coding mutation‐based ICP also helps identify the underlying mechanism of immune editing during immunotherapy. We found that the general trend of ctDNA kinetics for each involved gene is almost parallel, strongly suggesting a broad spectrum of T‐cell‐mediated attack associated with ICB immunotherapies, instead of targeting a few “key” epitopes in the anti‐tumor response. However, selective clonal evolution and subsequent acquired resistance during treatment have long been a concern, especially in the era of targeted therapies.31 ImmOne limitation of ICP‐based neoantigen tracking during immunotherapy probably lies in the pipeline for the in silico prediction of neoantigens. Current algorithms predict the peptide‐MHC binding affinity but cannot accurately predict proteasomal processing, post‐proteasomal trimming, and whether sufficient peptide‐MHC complexes reach the cell surface. Therefore, all neoantigen prediction strategies only provide a putative list with the greatest possibility to cover most bona fide tumor‐expressing neoantigens. Furthermore, the lead time for the clinical application of ICP, including WES, neoantigen prediction, and the synthesis of patient‐specific sequencing probes, constrains our ability to make real‐time neoantigen tracking observations in the first two cycles of ICB administration. Presently, this study was limited in its cohort size and was performed for feasibility assessment purposes, partly due to the technical demands of the approach. The clinical significance of ICP‐based ctDNA sequencing needs to be validated with a larger cohort, with a prospective trial design and long‐term surveillance scans, after which, ICP‐based ctDNA sequencing could potentially improve our understanding of neoantigenic evolution and minimize unnecessary ICB‐mediated immunological toxicity, ultimately maximizing the clinical benefits of checkpoint blockade immunotherapies.4ICP‐based ctDNA sequencing provides a superior coverage to longitudinally track predicted dominant neoantigens. The clinical application of such an approach will warrant personalized management strategies that in turn improve the clinical benefits of immunotherapies and enhance our understanding of ICB‐driven tumor editing and neoantigenic profile evolution.5EGFR mutation or ALK‐EML4 fusion at Chongqing Xinqiao Hospital. Ten formalin‐fixed, paraffin‐embedded (FFPE) tumor tissues and matched peripheral blood samples were obtained prior to the first infusion of ICB. During the course of treatment, 97 time‐course blood samples (including baseline samples) were collected for ctDNA sequencing. Clinical responses were evaluated by a senior physician based on RECIST 1.1 criteria every six weeks (two cycles of ICB administration). Tumor burden was measured by the sum of the longest diameter of evaluable lesions according to RECIST 1.1 criteria (designated as RECIST‐measured tumor burden), or by the sum of the product of perpendicular diameter of evaluable lesions (designated as SPD‐measured tumor burden). The study protocol was approved by the institution review board of Xinqiao Hospital, Army Medical University. Written informed consent for sample acquisition was obtained from all patients. All data were deidentified.Ten patients diagnosed with stage IIIB/IV non‐small cell lung cancer (NSCLC) were treated with durvalumab monotherapy or combination therapy with tremelimumab/durvalumab . All patients were confirmed to lack an Genomic DNA (gDNA) was extracted from the FFPE samples using the GeneRead DNA FFPE Kit  and from peripheral blood mononuclear cells using the DNA Blood Midi/Mini kit (Qiagen) according to the manufacturer's instructions. Plasma cell‐free DNA (cfDNA) was isolated using the MagMAX Cell‐Free DNA Isolation Kit  according to the manufacturer's protocol. The quality of purified DNA was assayed by gel electrophoresis and quantified with a Qubit 4.0 Fluorometer . In‐house panels were designed to capture cfDNA fragments to generate sequencing libraries. The sequencing libraries were evaluated using the NovaSeq 6000 platform  in 150PE mode.Purified gDNA was first fragmented into DNA pieces of approximately 300 bp using an enzymatic method . After end‐repairing and A tailing, T‐adaptors were ligated on both ends, followed by PCR amplification to obtain a pre‐library. The final sequencing libraries were prepared using the 96 rxn xGen Exome Research Panel v1.0  according to the manufacturer's protocol. For the targeted sequencing of cfDNA, the pre‐libraries were prepared according to the method previously described.37 In‐ The FASTQ files were aligned to the human reference genome (hg19/GRCh37) using Burrows–Wheeler Aligner . Picard (2.12.1) (https://picard.sourceforge.net/) was used to process PCR duplicates for mapped BAM files. GATK  was used for local realignment and base quality recalibration was employed to compute sequencing coverage and depth. Single nucleotide variants (SNVs) and small insertions and deletions were identified using GATK MuTect2. Mutations in the ENCODE Data Analysis Consortium blacklist were removed.The raw sequencing reads were subjected to quality control by trimming adaptor sequences and removing poly‐N sequences (>10%) and low‐quality reads (<Q20) preprocessed using FASTP.38 The based on multiple databases, including HGVS variant description and population frequency databases , disease or phenotype databases , and variant functional in silico prediction tools (PolyPhen‐2 and SIFT). After annotation, SNVs annotated as genomicSuperDups with a variant allele frequency (VAF) < 0.2 or PopFreqMax > 0.05 were excluded and nonsynonymous SNVs with a VAF > 3% or with a VAF > 1% in cancer hotspots collected from patient databases were retained for further analyses.Variants were annotated using ANNOVAR41 bas We applied a binomial test to compute the 95% confidence interval (CI) of VAF, which was used to calculate the 95% CI of CCF thereafter. A mutation was regarded as subclonal if the upper boundary of the 95% CI of CCF was <1, and clonal otherwise. Tumor mutation burden was defined as the total number of non‐synonymous SNVs per megabase of coding sequence in a tumor genome based on WES.For each mutation, the proportion of mutated reads , the proportion of tumor cells harboring the mutation , and the clonality were calculated according to the methods described by Letouzé et al.40 We  to infer the individual HLA type and pVAC‐Seq to predict the binding affinity of non‐silent mutant peptides. To reduce redundancy and select neoepitopes predicted to exhibit strong specific HLA‐peptide binding, 500 nm MHC binding affinity was applied as a filtering criterion. The predicted neoantigens were sorted by their binding score. For individual patients with more than 20 predicted neoantigens, the neoantigens with the top 30 binding scores were selected to design DNA probes to assay their presence and abundance in plasma during ICB treatment. For individual patients with fewer than 20 predicted neoantigens, all predicted neoantigens were selected, supplemented with mutations with the highest VAFs to obtain a total of 20 loci.Dominant neoantigens were predicted using two computational tools, OptiType42 to  was used to trim adapters and to remove low‐quality sequences to obtain clean reads. The clean reads were aligned against the Ensemble GRCh37/hg19 reference genome using BWA. PCR duplicates were processed using gencore, and consensus reads were generated. SAMtools was applied for the detection of SNVs, insertions, and deletions. HGVS variants were annotated using ANNOVAR.FASTP38 wasp‐values were adjusted based on the Benjamini–Hochberg correction.Statistical analyses and data visualization were conducted using R/Bioconductor packages. The Kaplan–Meier method was used for the survival analysis, median values were compared using log rank tests, and hazard ratios were obtained from the Cox proportional hazards model. Overlapping genes between ICP, WES, and commercially available panels were evaluated by Kruskal–Wallis tests. Dunn's test was used for post‐hoc analyses, and The authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supporting TableS1Click here for additional data file.Supporting TableS2Click here for additional data file.Supporting TableS3Click here for additional data file.Supporting TableS4Click here for additional data file.Supporting TableS5Click here for additional data file.


Dear Editor,PTPRT) has been found to be the most frequently mutated PTP gene in cancers and could predict poor prognosis;PTPRT mutation with clinical outcomes of ICI remains unknown. Here, we performed a comprehensive pancancer investigation to clinically validate PTPRT mutation as a predictive biomarker for ICI therapy.We and others have shown that the tumor mutation burden (TMB) and several underlying oncogenic alterations could provide clinically predictive implications for immune checkpoint inhibitor (ICI).PTPRT mutational data quantified by whole exome sequencing of 2129 cancer patients treated with ICI and 10,814 cancer patients without receiving ICI from the cBioPortal, PubMed, and The Cancer Genome Atlas. The study protocol was approved by the ethics committee of the Sun Yat‐sen Memorial Hospital of Sun Yat‐sen University. The requirement for informed consent of study participants and the permission to use the patient data were waived because the human data were obtained from publicly available datasets. All analyses were performed according to the STROBE guideline from September 18 through October 1, 2019. Overall survival (OS) were primary outcomes, which were computed using the Kaplan‐Meier method and were assessed with the log‐rank test and the hazard ratio (HR) calculated by the Cox regression model. The TMB in PTPRT wild‐type versus mutant groups were compared with Wilcoxon rank‐sum tests. All analyses were performed using R (version 3.4.4) and were considered statistically significant if P values < .05.We collected clinical and PTPRT mutant; Figure PTPRT mutant group versus PTPRT wild‐type group  out of 10,814 patients without receiving ICI across 33 cancer types, among which the mutation frequency was 28.4% in melanoma, 11.1% in esophagogastric adenocarcinoma, 10.9% in endometrial carcinoma, 8.6% in colorectal adenocarcinoma, and 8.0% in NSCLC .PTPRT mutation in patients without receiving ICI. Among 10,814 pancancer patients, there was no difference in OS between PTPRT mutant and wild‐type  and 431 melanoma patients (126 [29.2%] PTPRT mutant), no difference in OS between PTPRT mutant and wild‐type patients was observed, either  and FDA‐approved FoundationOne CDx (F1CDx). Therefore, PTPRT should be considered together with other known essential genes to expand the landscape of immuno‐oncological genomic panel, and should be integrated into multiomics to more fully realize the precision immunotherapy. In‐deep characterization of PTP expression pattern could be informative for understanding patterns of immune escape and the selection of candidates for immunotherapy.To the best of our knowledge, this is the first study to identify the mutation status of PTPRT, since PTPRT mutation was demonstrated to be promisingly predictive of immunotherapy efficacy in our study and has been found to determine bevacizumab resistance in the study conducted by Hsu et el.PTPRT mutation and ICI. Future prospective trials with a larger sample size, more detailed clinical treatment information and a longer follow‐up are needed to validate the pancancer applicability of PTPRT mutation status and in‐deep characterize how PTPRT mutation interact with immune system to influence ICI benefit.Moreover, PD‐L1 inhibitor atezolizumab plus VGFR inhibitor bevacizumab plus platinum‐based chemotherapy was shown to have an encouraging survival benefit in recent randomized IMpower 150 trial.PTPRT mutation status could serve as a predictive biomarker for ICI in pancancer and specifically in NSCLC and melanoma.In conclusion, 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.The study protocol was approved by the ethics committee of the Sun Yat‐sen Memorial Hospital of Sun Yat‐sen University. The requirement for informed consent of study participants and the permission to use the patient data were waived because the human data were obtained from publicly available datasets.