A typical neuron consists of a soma, a single axon with numerous nerve terminals, and multiple dendritic trunks with numerous branches. Each of the 100 billion neurons in the brain has on average 7,000 synaptic connections to other neurons. The neuronal endolysosomal compartments for the degradation of axonal and dendritic waste are located in the soma region. That means that all autophagosomal and endosomal cargos from 7,000 synaptic connections must be transported to the soma region for degradation. For that reason, neuronal endolysosomal degradation is an extraordinarily demanding and dynamic event, and thus is highly susceptible to many pathological conditions. Dysfunction in the endolysosomal trafficking pathways occurs in virtually all neurodegenerative diseases. Most lysosomal storage disorders (LSDs) with defects in the endolysosomal system preferentially affect the central nervous system (CNS). Recently, significant progress has been made in understanding the role that the endolysosomal trafficking pathways play after brain ischemia. Brain ischemia damages the membrane fusion machinery co-operated by N-ethylmaleimide sensitive factor (NSF), soluble NSF attachment protein (SNAP), and soluble NSF attachment protein receptors (SNAREs), thus interrupting the membrane-to-membrane fusion between the late endosome and terminal lysosome. This interruption obstructs all incoming traffic. Consequently, both the size and number of endolysosomal structures, autophagosomes, early endosomes, and intra-neuronal protein aggregates are increased extensively in post-ischemic neurons. This cascade of events eventually damages the endolysosomal structures to release hydrolases leading to ischemic brain injury. Gene knockout and selective inhibition of key endolysosomal cathepsins protects the brain from ischemic injury. This review aims to provide an update of the current knowledge, future research directions, and the clinical implications regarding the critical role of the neuronal endolysosomal trafficking pathways in ischemic brain injury. The endolysosomal system is a major catabolic pathway for recycling cellular waste products. Newer evidence has placed this system at the center stage of many research fields. This system has previously been viewed as a relatively static acidic compartment containing hydrolytic enzymes for cellular waste degradation, but now has emerged as a highly dynamic organelle capable of sensing and controlling multiple cellular processes  . The M6P-tagged procathepsins are recognized and bound by M6P-receptors (M6PR), packed together into clathrin-coated vesicles, and transported to the late endosomes  domains termed D1 and D2. The D2 domain has slow adenosine triphosphate (ATP)-hydrolytic activity to maintain the NSF hexameric structure. The D1 domain is thought to be the main engine that uses ATP hydrolysis for SNARE complex disassembly and reactivation .Sophisticated neuronal waste transport systems are required to cope with the long-distance trafficking from distal synaptic connections to the soma region where the endolysosomal degradation compartments are located  and endovascular treatment may improve clinical outcomes. However, only 5–10% of stroke patients are eligible for these reperfusion therapies. Furthermore, these reperfusion therapies rely on removing vascular blockage and can inadvertently lead to an ischemia-reperfusion injury. At present, there exist no therapies directed toward brain ischemia-reperfusion injuries.Smith et al.  found thDeficiency in the endolysosomal system is observed virtually in all neurodegenerative disorders and in a significant number of LSDs  focal brain ischemia caused a large-scale release of CTSB that damaged all neuronal structures indiscriminately. As a result, the post-ischemic neurons rupture, releasing their contents which include CTSB, ubiquitinated protein aggregates, and p62-protein aggregates into the extracellular space.Yuan et al.  showed tAs described above, the depletion or disappearance of intra-neuronal NSF immunoreactivity was mainly due to NSF deposition into a detergent (Triton X100)/salt-insoluble aggregates after both global and focal brain ischemia  requires binding to ATP or non-hydrolysable ATP analogs to maintain its active soluble symmetrical homohexamer conformation . The increased levels of LC3-II and p62/SQSTM1 often indicate that autophagic flux is disrupted or autophagic degradation activity is reduced , endosomal marker proteins (syntaxin13 and EEA1), autophagic marker proteins (LC3II and p62), and endolysosomal marker proteins (LAMP1/3 and CTSB)  CTSB, TGN38, p62, and ubiquitin immunoreactivities that were normally located inside sham-operated control neurons, were found abnormally distributed outsides of neuronal soma in the brain region where selective neuronal death occurs after brain ischemia –5 Yuan ; (ii) ceN-ethylmaleimide sensitive factor depletion leads to an ever-increasing buildup of endolysosomal compartments. A prolonged buildup of endolysosomal compartments leads to endolysosomal membrane damage or rupture, leading to the release of cathepsins  grants: NS36810, NS40407, NS097875, and NS102815 to BH; by Veteran Affair Merit grant: I01BX001696; and by American Heart Association 0940042N-5 to BH.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Clonal evolution represents the natural process through which cancer cells continuously search for phenotypic advantages that enable them to develop and expand within microenvironmental constraints. In chronic lymphocytic leukemia (CLL), clonal evolution underpins leukemic progression and therapeutic resistance, with differences in clonal evolutionary dynamics accounting for its characteristically diverse clinical course. The past few years have witnessed profound changes in our understanding of CLL clonal evolution, facilitated by a maturing definition of high-risk CLL and an increasing sophistication of next-generation sequencing technology. In this review, we offer a modern perspective on clonal evolution of high-risk CLL, highlighting recent discoveries, paradigm shifts and unresolved questions. We appraise recent advances in our understanding of the molecular basis of CLL clonal evolution, focusing on the genetic and non-genetic sources of intratumoral heterogeneity, as well as tumor-immune dynamics. We review the technological innovations, particularly in single-cell technology, which have fostered these advances and represent essential tools for future discoveries. In addition, we discuss clonal evolution within several contexts of particular relevance to contemporary clinical practice, including the settings of therapeutic resistance to CLL targeted therapy and immunotherapy, as well as Richter transformation of CLL to high-grade lymphoma. Clonal heterogeneity and evolution are among the most fundamental properties of cancer. Through a reiterative process of clonal proliferation, diversification and Darwinian selection, cancers continually adapt within the host microenvironment, progressively acquiring and accumulating enabling attributes that allow them to develop and expand . Intratu+ CD5+ B lymphocytes, offers an informative disease model to study cancer evolution. First, CLL is characterized by clinical heterogeneity that encompasses a range of disease trajectories including rapid progression, treatment refractoriness and high-grade transformation at one end of the spectrum (Chronic lymphocytic leukemia (CLL), a malignancy of CD19spectrum , 5, to aspectrum , 7. Thisspectrum , 9. Tumospectrum .The clinical heterogeneity of CLL necessitates identification of biological correlates of high-risk CLL, in order to define the patient population most at risk of CLL progression that merits close monitoring and focused study. Over the past two decades, biomarkers of high-risk CLL have evolved with our increasingly sophisticated understanding of CLL biology. Concurrently, advances in bulk sequencing and more recently integrative single-cell sequencing technology have facilitated the longitudinal study of CLL clonal evolution in this patient group. These studies have illuminated our understanding of CLL clonal architecture and the complex clonal evolutionary dynamics that give rise to CLL progression, resistance to different CLL treatments and high-grade transformation, revealing diverse biological processes and novel mechanisms. In this review, we will appraise recent advances in our understanding of intratumoral heterogeneity and clonal evolution in patients with high-risk CLL, and discuss the technological innovations that have facilitated this understanding. We will focus particularly on clonal evolution within several topical contexts, including the settings of therapeutic resistance to CLL targeted therapy and immunotherapy, as well as Richter transformation of CLL to high-grade lymphoma.Our understanding of what constitutes high-risk CLL has evolved considerably over recent years (-mutated counterpart (M-CLL)  , 16, rel (M-CLL) , 18. BCR (M-CLL) –22. Nota (M-CLL) . Recent mutation . Moreovein-situ hybridization (FISH) has enabled CLL risk stratification into distinct cytogenetic risk categories with del(17p) carrying the highest risk, del(11q) and trisomy 12/normal FISH conferring high and intermediate risk respectively, and isolated del(13q14) being associated with lower risk   . FinallyCLL-IPI) –31.SF3B1 and NOTCH1 mutations, identified patients at higher risk of disease progression as well as disease recurrence after chemotherapy-based CLL therapy  with prognostic significance, each corresponding to a distinct transcriptional profile that reflects a unique CLL phenotypic state . On thisSome of these newer biological correlates of high-risk CLL, such as adverse epitypes and ECs as well as high epiCMIT, require further prospective validation. Nevertheless, their characterization has offered opportunity for the integration of genetic, transcriptional, epigenetic, phenotypic and clinical parameters to refine prognostic stratification, thereby providing a more accurate definition of high-risk CLL. Indeed, a recent multicenter effort by Knisbacher and colleagues utilizing data acquired from hundreds of patient samples have generated multivariate prognostic models that incorporate these different parameters . While mThe clinical heterogeneity of CLL is quite evident in highly disparate clinical trajectories that can be observed amongst patients. An indolent or slowly progressing clinical course is observed in the majority of CLL patients, with rapid disease progression and spontaneous regression at the two extremes. These different clinical trajectories are mirrored by differences in clonal growth dynamics . Growth Through the analysis of serial samples from CLL patients, Gruber and colleagues linked differences in CLL growth trajectories to variations in clonal genetic composition as well as the extent of clonal evolution . CompareThe study of clonal evolution in cancer is reliant on data generation and analytical platforms that are capable of delineating clonal architecture and subclonal phylogenetic relationships from longitudinal patient samples , 45. In MYD88 mutation, trisomy 12 and del(13q14), reflect early genomic events that mediate CLL development. On the other hand, other driver mutations that are typically present in only a fraction of CLL cells are subclonal and likely represent later events that arise from subclonal selection. Subclonal mutations are thought to confer enhanced clonal fitness and drive CLL progression  demonstrate global transcriptional changes that often accompany genetic clonal evolution  of each driver that corrects for CLL sample purity and chromosomal copy number alteration , 44. Anagression , 44. Exavolution –48, whilvolution .Despite their proven utility, a fundamental limitation of clonal evolution studies based on bulk sequencing methodologies lies in their inability to resolve with precision subclonal phylogenetic relationships at low CCFs, because the capacity to detect rare subclonal genomic events is often limited by sequencing read depth. Moreover, bulk analyses do not readily permit an integrative analysis of the genetic, epigenetic and transcriptional dynamics of individual CLL subclones that is essential to understand complex evolutionary mechanisms. These limitations can be addressed through contemporary approaches that leverage multidimensional single-cell sequencing technology.in vitro and in vivo CLL models  , 52, andMitochondrial DNA heteroplasmy are naturally occurring, stochastic mtDNA mutations that can serve to identify individual tumor subclones . Such muSimilar to mtDNA mutations, stochastic DNA methylation changes known as epimutations are heritable marks that can be adopted for lineage tracing . Epimutain vitro and in vivo models to interrogate CLL clonal evolution. An example of this is ClonMapper that enforces expression of unique single-guide RNA (sgRNA) barcodes within single cells  class I and II immunopeptidomes, complemented by computational analysis of genomic and transcriptomic data , 63, enaAltogether, these exciting new technological innovations will undoubtedly further advance our understanding of clonal evolution in high-risk CLL. In order to appreciate the context upon which future discoveries can be made, our current understanding of the molecular basis of intratumoral heterogeneity and clonal evolution in CLL is reviewed in the next sections.TP53 mutations in their CLL clone provide further testament to the notion that the mere presence of CLL genetic drivers is insufficient to drive clonal evolution or disease progression , DNA damage response  (POT1 mutation)  , 44, 71.utation)  or telomutation) , giving Clonal evolution studies based on the analysis of WES or WGS data have enabled the characterization of a founder clone in CLL patients wherein somatic mutations are present , 64. Thede novo CLL-like disease with distinct IGHV rearrangements upon xenotransplantation into immunodeficient mice  or different drivers (‘divergent evolution’) to compete for dominance.Clonal evolution, on the other hand, can be considered from a purely genetic standpoint as the natural consequence of differential growth accelerations conferred by the different genetic drivers within competing subclones. In CLL, the strongest growth accelerations appear to be associated with tumor suppressor genes such as leration . Differeleration . The hetCLL treatment has consistently been shown to fuel clonal evolution, resulting in fitter subclones with higher growth rates at the time of relapse. The resultant evolutionary pattern can be seen as a function of the pre-existing subclonal landscape at the time of treatment, and the nature of that CLL treatment. The importance of the former is evidenced from observations that a CLL harboring a wider spectrum of genetic alterations, more subclonal drivers, more complex subclonal landscapes, and exponential growth at the time of treatment is likely to experience more profound clonal shifts in response to treatment , 44. ThiTP53-mutant and/or del(17p) CLL subclones are almost invariably selected under chemotherapy-based treatments  and BCL-2 inhibitors (e.g. venetoclax) are increasingly replacing conventional chemotherapy-based treatments –127. AltBTK) or PLCG2 genes  relapsing from venetoclax in a recent study by Blombery and colleagues (BTK/PLCG2 or BCL2 mutations remain at very low CCFs (<0.1) at the time of relapse  in ibrutinib-resistant cases, the presence of additional alterations appears to be a prerequisite for the selection of subclones harboring CDKN2A/B and BTG1 defects with venetoclax. Finally, Guièze and colleagues reported amp(1q) as a recurrent lesion in a subset of venetoclax-resistant patients with wild-type BCL2  pathway  is predictive of long-term treatment outcome within various therapeutic settings –149, andAllogeneic HSCT exemplifies the power of harnessing antitumor immunity for cancer treatment, but relapses nevertheless occur. Recently, Bachireddy and colleagues investigated the evolutionary dynamics underlying resistance to graft-versus-leukemia (GvL) effect, uncovering mechanisms that may have potential broader relevance for CLL immunotherapy . ThroughAs apparent from the above, the pattern of clonal evolution leading to relapse post allo-HSCT differs markedly from the evolutionary dynamics that mediate resistance to chemotherapy or targeted therapy. This serves to illustrate the vastly different selection pressures that are imposed by different treatment modalities. The work of Bachireddy et al. also exemplifies the use of integrative multidimensional approaches to dissect clonal evolution which allow us to appreciate the non-genetic and genetic determinants of clonal dynamics, a comprehensive understanding of which will enable us to devise better treatments to manage CLL relapse.Richter Syndrome (RS), characterized by the high-grade transformation of CLL to diffuse large B cell lymphoma (DLBCL) and less commonly to Hodgkin lymphoma, represents a catastrophic clinical sequel of high-risk CLL. RS occurs in an estimated 2% to 10% of CLL patients . IndividTP53 inactivation and/or CDKN2A/B loss, alongside MYC activation, and the second involving trisomy 12. In addition, NOTCH1 mutations were found frequently in patients with RS, particularly among individuals also harboring trisomy 12. The second study by Fabbri et al.  clinical lectureship. This work is supported by grants from the National Institutes of Health (NCI-1RO1CA155010), the NIH/National Cancer Institute (NIH/NCI)  and the CLL Global Research Foundation.CW holds equity in BioNTech, Inc. and receives research funding support from Pharmacyclics.The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The first involved analysis of data on virus aerosol dispersion in aircraft cabin mock-ups from a previous study conducted at KSU during July–August 2017 as part of a pandemic influenza research initiative , resultiIn November 2020, CDC applied this data-driven model to simulate the protective effect of a vacant middle seat versus full aircraft occupancy. Two analytical approaches were used. Both approaches analyzed reductions in relative exposures  rather than absolute exposure.The first approach considered only the extra distance between passengers created by the vacant middle seat. The regression model estimated exposure as a function of distance to assess the exposure reduction of moving an adjacent passenger one seat further away from an infectious passenger, leaving an empty middle seat between them. The distance effect was explored further to simulate the total exposure reduction for groups of passengers up to and including a full simulated cabin of 120 seats.The second approach combined the distance effect predicted by the regression model and the reduced occupancy effect predicted by simple probability estimation, as these are inseparable in realistic arrangements of infectious passengers and other passengers. When simply defining exposure risk as reduced occupancy, a vacant middle seat reduced exposure by an estimated 33% compared with full occupancy, in single-aisle, three-seats-per-side cabins, because there are 33% fewer potentially infectious passengers.The first approach predicted a 23% exposure reduction by moving an adjacent passenger one seat further away from an infectious passenger. The total reduction in relative exposure for a full 120-seat cabin yielded reduction of 35.0%–36.4%, 35.1%–38.2%, and 35.9%–39.4% for one, two, and three infectious passengers, respectively, depending on their seating pattern. All sources were placed in window or aisle seats such that the potential reduction in number of infectious passengers onboard from vacant middle seating was not considered . The secThis laboratory-based model predicts a 23% to 57% reduction in exposure to viable virus particles when middle seats on an airline are kept vacant. This range is comparable to that reported in another study that used computational fluid dynamics simulation and considered cabin ventilation rates per passenger to show that keeping middle seats vacant reduced the risk for airborne infection by 45%.Aircraft cabin environmental control systems (ventilation systems) are designed to deliver amounts of clean air per occupant that conform to various standards.It is important to recognize that the current study addresses only exposure and not transmission.2) values (the percentage of the response variable variation explained by a model) above 70%, suggesting that the distance model explained most of the observed virus concentration behavior. Third, the use of spray bottles to emit droplets, followed by 5 minutes of air sampling, might not fully represent the variety of respiratory events that could transmit virus . Mandated mask use further alters the human respiratory source, making the relative exposure approach used here an important way to diminish bias related to release volume. Finally, the study only assessed aerosols, not fomites and droplets. Exposures decrease more rapidly with distance for these exposure paths; therefore, distancing would have an even larger protective effect than that observed in this study.The findings in this report are subject to at least four limitations. First, data were collected under higher relative humidity conditions in the laboratory than would be present during flight. Droplet evaporation into aerosol is more rapid under lower relative humidity. Because aerosols travel farther than droplets, the current study might underpredict the aerosol spread in an actual cabin environment. The slower evaporation in the current study might then overpredict the observed effect of distancing because this more rapid decrease makes estimated distance effects larger. Second, in the data used to build the regression models, most of the variability was within approximately 5 ft of the infection source. Although this near-zone variability weakens the quantification of the effect of short distances, the equations were the statistical best fit and had coefficient of determination (RBased on a data-driven model, approaches to physical distancing, including keeping middle seats vacant, could reduce exposure to SARS-CoV-2 on aircraft. The extent to which exposure reduction might decrease transmission risk is not yet understood. Current CDC guidelines recommend against travel for persons who have not been vaccinated and require masking for all persons while on aircraft. Physical distancing of aircraft passengers, including through policies such as middle seat vacancy, could provide additional reductions in SARS-COV-2 exposure risk. This study could help inform future modeling of transmission risk, which might encompass determinants that were not fully explored here such as mask use, virus characteristics, and host characteristics, such as vaccination status.Aircraft can hold large numbers of persons in close proximity for long periods, which are conditions that can increase the risk for transmitting infectious diseases.Based on laboratory modeling of exposure to SARS-CoV-2 on single-aisle and twin-aisle aircraft, exposures in scenarios in which the middle seat was vacant were reduced by 23% to 57%, compared with full aircraft occupancy, depending upon the model.Physical distancing of airplane passengers, including through policies such as middle seat vacancy, could provide additional reductions in risk for exposure to SARS-CoV-2 on aircraft.


This Commentary is in response to the BMC Urology publication entitled “Infection-related hospitalization following ureteroscopic stone treatment: Results from a surgical collaborative”. This study utilized a registry with prospectively recorded standardized data elements named Reducing Operative Complications from Kidney Stones, part of the Michigan Urological Surgery Improvement Collaborative, to identify risk factors of infection-related hospitalization after ureteroscopy for stone treatment. The study included 1817 primary URS procedures for urinary stones in 11 practices in Michigan. They found 43 patients (2.4%) were hospitalized with an infection-related complication and 3 patients died during their hospitalization . Just over 20% of patients did not have a pre-operative urinalysis or urine culture, representing a deviation from guideline recommendations. Also, in the hospitalized group, none of the 12 patients (27.9%) who had a positive pre-operative urinalysis or urine culture received pre-operative treatment. A multivariable analysis identified higher Charleston Comorbidity Index, history of recurrent urinary tract infection, increasing stone size, intraoperative complications, and fragments left in-situ as independent risk factors for hospitalization from an infection after ureteroscopy. This commentary discusses caveats to the data as well as short-comings of the study. It also reviews more broadly infection after ureteroscopy, includes findings from similar studies, and highlights guideline recommendations to reduce infection risk. Commentary:A systematic review from the European Association of Urology (EAU) Section of Urolithiasis including over 24,000 ureteroscopy (URS) procedures from 14 large studies found an overall infectious complication rate of 3.9% [The Michigan Urological Surgery Improvement Collaborative (MUSIC) is a physician-led quality improvement (QI) collaborative that includes academic, private practice, and community urologists across the state of Michigan . Their wIn the current study, the authors used the ROCKS registry to identify 1817 URS procedures in 1737 patients undergoing primary URS for urinary stones in 11 practices in Michigan . AbstracA multivariable analysis identified higher Charleston Comorbidity Index, history of recurrent urinary tract infection (UTI), increasing stone size, intraoperative complications, and lithotripsy with fragments left in-situ as independent risk factors for hospitalization from an infection-related complication after URS. The authors appropriately recognize the limitations of an extensive multivariable analysis with a small number of events. Pre-operative alpha blockade was also identified as a factor decreasing the risk of hospitalization from infection-related complications; however, this did not hold true on multivariable analysis. The reason for the alpha-blocker therapy was not recorded so this could represent a lower risk group who were more likely to be followed on medical expulsive therapy. This is just one example of the types of confounding variables present in analyses such as these.There are several important caveats to the data presented in this study. Patients were over 18 years of age undergoing unilateral URS for urinary stones. Anyone with an ipsilateral nephrostomy at the time of URS, or who underwent URS after percutaneous renal surgery were excluded. Thus, the results are not generalizable to these groups of patients, pediatric patients, or patients undergoing bilateral URS. Also, hospitalization for infection-related complications was identified through retrospective chart review based on systemic inflammatory response (SIRS) criteria. The causes for admission due to infection after URS can be variable and the authors appropriately recognize that more patients may have been hospitalized without SIRS criteria that were not captured. Furthermore, this analysis does not endeavor to address the broader issue of infection risk after URS including those instances that do not result in hospitalization but cause an increase in phone encounters and office visits.The authors also appropriately recognize the inherent limitations of the analysis based on the registry data collection tool. For example, the collection tool did not capture data on the type of laser lithotripsy technique utilized. Thus patients undergoing a “dusting” technique would have likely been coded as having fragments left in-situ, regardless of the size of fragments. This could make “fragments left in-situ” a surrogate for stone size. Also, information on stone cultures was not collected. Stone cultures have been shown to be better predictors of sepsis and SIRS than voided cultures , 7. EswaLimitations aside, this study revealed that over 20% of their patients undergoing URS did not have either a pre-operative urinalysis or urine culture (PUC). This is a surprisingly prevalent deviation from the standard of care as recommended in both the AUA Guidelines on Surgical Management of Stones and the EAU Guidelines on Interventional Treatment for Urolithiasis , 11. CarThe current study reinforces many previously described risk factors and predictors for hospitalization from infectious complications after URS in a diverse cross-section of patients and practice environments. In the aforementioned EAU systematic review, the most commonly identified risk factors were higher comorbidity scores, recurrent UTIs, larger stones, and positive PUCs , echoingThe EAU catalogued risk modification strategies suggested by the various manuscripts included in their systematic review. Some strategies suggested include the appropriate use of antibiotic prophylaxis, broadened prophylaxis for pre-stented patients and longer operations, utilization of local resistance patterns when choosing antibiotics, and improved pre-operative counseling. In another review of infectious complications in stone disease, Wollin et al. highlighted the importance of identifying high-risk patients, treating active UTIs pre-procedure, and ensuring a negative PUC . All of Although there are many studies in the literature examining risk factors and predictors for infectious complications after URS for stone disease, most are single institution series and/or from academic medical centers which limits the generalizability of the results. The prospectively captured data from the current study include a diverse patient population and represent a variety of practice environments and practitioners, thus, making these findings more relatable and relevant. The work being done by MUSIC and ROCKS is truly important work and the authors are trailblazing in urology QI in the United States.


Hereditary leiomyomatosis and renal cell carcinoma (HL (RCC)) entails cutaneous and uterine leiomyomatosis with aggressive type 2 papillary RCC-like histology. HLRCC is caused by pathogenic variants in the FH gene, which encodes fumarate hydratase (FH). Here, we describe an episode of young-onset RCC caused by a genomic FH deletion that was diagnosed via clinical sequencing. A 35-year-old woman was diagnosed with RCC and multiple metastases: histopathological analyses supported a diagnosis of FH-deficient RCC. Although the patient had neither skin tumors nor a family history of HLRCC, an aggressive clinical course at her age and pathological diagnosis of FH-deficient RCC suggested a germline FH variant. After counseling, the patient provided written informed consent for germline genetic testing. She was simultaneously subjected to paired tumor profiling tests targeting the exome to identify a therapeutic target. Although conventional germline sequencing did not detect FH variants, exome sequencing revealed a heterozygous germline FH deletion. As such, paired tumor profiling, not conventional sequencing, was required to identify this genetic deletion. RCC caused by a germline FH deletion has hitherto not been described in Japan, and the FH deletion detected in this patient was presumed to be of maternal European origin. Although the genotype-phenotype correlation in HLRCC-related tumors is unclear, the patient’s family was advised to undergo genetic counseling to consider additional RCC screening. Hereditary leiomyomatosis and renal cell carcinoma (HL(RCC)) is an inherited syndrome that causes multiple cutaneous leiomyomas, severely symptomatic uterine leiomyomas, and papillary type 2 RCC-like histology owing to anomalies in the FH gene that encodes fumarate hydratase (FH). The severity of HLRCC varies significantly within and between families. Although the penetrance of HLRCC-related RCC is not considered high, the prognoses of patients with this progressive condition are reportedly poor. For example, 9 of 13 patients in a particular study reportedly died of renal cancer metastasis within five years of their diagnosis [Smit et al. reported that the penetrance of cutaneous leiomyoma in their cohort was almost 100% after age 40 and that 11 of 21 women who were FH pathogenic variant carriers (52%) were treated for uterine myomas by the age of 35 years [The FH gene controls energy metabolism, and FH is involved in the tricarboxylic acid (TCA) cycle in mitochondria . The enzIn FH-deficient RCC, oxidative phosphorylation is impaired, and aerobic glycolysis is upregulated (a phenomenon referred to as the Warburg effect). Energy production in cancer cells thereby becomes almost fully dependent on glycolysis, the increase of which amplifies adenosine triphosphate production, which in turn promotes cell proliferation. Through this mechanism, VEGF (promoting angiogenesis) and GLUT1 (increasing glucose transport) are upregulated. Both these events underlie the molecular mechanism of high-grade RCC . FH-defiBRCA1/2 and Lynch syndrome-causative genes. The European Society of Medical Oncology (ESMO) Precision Medicine Working Group recommended germline-focused analysis of tumor-only sequences in 2019, and FH was included among the 27 targeted genes [FH is not included in the list of genomic “secondary findings” devised by the American College of Medical Genetics and Genomics (ACMG) in 2017 . The oried genes . At KeioMolecular tumor profiling is an epochal method for determining therapeutic targets. Precision medicine has recently been advocated, and personalized treatment strategies based on tumor profiling have attracted attention. As a concern for tumor profiling, the possibility of hereditary cancer-related genes can be confirmed by comparison with paired normal tissues as controls. The ACMG (2020) issued a statement on presumed germline pathogenic variants that can be revealed by tumor profiling tests . They stBy disclosing these germline findings, useful information can be provided to patients and their families, appropriate surveillance methods for early detection and early treatment for cancers can be suggested, indications for risk-reducing surgery can be discussed, and appropriate treatment for individual patients can be selected .Here, we describe a patient with early-onset RCC caused by a genomic FH deletion that was diagnosed via tumor profiling analysis.A 35-year-old woman with no previous history of disease was diagnosed with RCC and multiple metastases while undergoing examination for abdominal pain. Renal biopsy was performed, and histopathological analyses supported the diagnosis of FH-deficient RCC .The patient was diagnosed with stage-4 RCC as well as multiple metastases in the lung, liver, supraclavicular lymph nodes, para-aortic lymph nodes, adrenal glands, and retroperitoneum. Combination therapy with ipilimumab and nivolumab (a standard treatment for RCC) was started immediately after diagnosis. After three doses of the combination therapy, the symptoms worsened, marked accumulation of ascites was noted, and cancer-related pain was exacerbated. Hence, the patient was subjected to a PleSSision Exome test to determine the therapeutic target.Although the patient had neither skin tumors nor a family history of HLRCC , an aggrAfter genetic counseling, written informed consent for germline genetic testing was obtained from the patient. However, at the time of tumor profiling, her RCC was already advanced and rapidly progressing, and she eventually died five months after diagnosis and four months after the start of treatment. Her death occurred before the test results were available; the large FH deletions found in the germline were disclosed to her family.Although conventional germline sequencing did not detect pathogenic FH variants, exome sequencing for tumor profiling revealed a heterozygous germline genetic deletion of FH. As PleSSision Exome test is a tumor-normal paired profiling test, genomic deoxyribonucleic acid (DNA) was extracted and purified from tumor tissues as well as peripheral blood mononuclear cells (PBMCs) obtained from the patient. Exome sequencing can detect not only single nucleotide variants (SNVs) but also copy number variants (CNVs), enabling a wide range of diagnoses. A large homozygous deletion of approximately 3 Mb near FH was confirmed in the tumor sample near the end of chromosome 1q as observed using the copy number alteration plot of the tumor-normal paired test . As HLRCWhen the copy number was tested in another panel for confirmation, results showed that the FH gene was not expressed at all in the tumor; there was a homozygous FH deletion at copy number = 0 in the tumor, and a heterozygous FH deletion, observed as copy number = 1 in the germline .When re-examining the deleted region in the tumor-normal paired exome analysis results in detail, the loss in CNV was detected to be limited to the FH gene’s vicinity. However, the variant allele frequency (VAF) plot showed that the loss of heterozygosity (LOH) or acquired uniparental disomy (UPD) might have occurred in the entire 1q chromosome in the tumor. In other words, the VAF was split more widely in chromosome 1q, suggesting that a LOH or acquired UPD had occurred in the tumor. Among the regions where the VAF plot split, three suspected deletions were found in the CNV  .These data led to the postulate that there were originally three LOH regions in the germline FH-deficient allele. In the process of carcinogenesis, a large deletion occurred in the healthy allele, and by copying the FH-deficient allele, 3 LOH regions appeared in both alleles in the cancerous tissue cells, resulting in acquired somatic UPD .EXO1 gene was also found to be deleted; this was consistent with the tumor profiling data  method, extensive germline FH deletion was confirmed, leading to the diagnosis of HLRCC a. Moreoving data b. These Immunohistochemical analysis was performed on formalin-fixed paraffin-embedded tissues using the FH mouse monoclonal antibody . In the PleSSision Exome test, genomic DNA was extracted and purified from tumor tissues and PBMCs obtained from the patient. Gene expression profiling was performed using the MiSeq sequencing platform (Illumina). Genome annotation and curation for analyzing the sequencing data were performed using the original bioinformatics pipeline GenomeJack  as previously described , and GRCAlthough large FH deletions have previously been reported, they are relatively rare in HLRCC. In a review of patients registered in the Leiden Open Variation Database (LOVD) published in 2008 , 93 of 1difference in the gene expression level rather than the position of the FH variant. Most variants are missense, nonsense, and frameshift mutations, but four percent are large deletions that can only be detected by MLPA [HLRCC disease onset and fumarase deficiency are more likely to be associated owing to the  by MLPA . Such laWe re-investigated the updated LOVD  and examIn our patient, a large deletion could not be detected (HLRCC may have been overlooked) when using conventional sequencing methods. Using the MLPA method first described in France in 2008, 19 patients who had previously been clinically diagnosed with HLRCC but had no pathological variants detected using direct sequencing were re-examined . An exonhttps://mgend.med.kyoto-u.ac.jp/mgend.med.kyoto-u.ac.jp, accessed on 26 July 2021.), which is a ClinVar-type, open-access database for use by the broad scientific community.Several previously reported Japanese families with HLRCC exhibited different pathogenic variants, including missense variants and a splice site variant that had not been reported in Caucasian families ,21,22,23Although we could not administer a genetic test to the patient’s younger brother because he lived abroad, we provided the family a letter with genetic test results and surveillance recommendations. Smit et al. reported that 100% of FH pathogenic variant carriers over the age of 40 years had cutaneous leiomyomas . In a prFH-related RCC is known to have a poor prognosis, as was the case for our patient. Muller et al. found that the lifetime risk of developing RCC in French FH pathogenic variant carriers is 19% . In 2011Several treatments, including mTOR inhibitors, are available for the treatment of FH-VAF-deficient RCC, but evidence of their effectiveness is insufficient . ConsideThe germline generic FH deletion in our patient was detected not via conventional sequencing but paired tumor profiling. RCC caused by a germline FH gene deletion has hitherto not been described in Japan, and FH deletion detected in this patient was presumed to be of maternal European origin. Although the genotype-phenotype correlation in patients with HLRCC-related tumors is unclear, the patient’s family was advised to undergo genetic counseling regarding HLRCC for further screening of renal malignancies. If there are no known patients with HLRCC in a family, the diagnosis of type 2 papillary RCC and/or FH-deficient RCC before the age of 40 should raise suspicions of this familial pathogenic variant. Finally, recent significant advances in screening technologies have improved the detection of large deletions upon genetic profiling of tumors; however, it is necessary to know the limits of each testing method when making a genetic diagnosis.


Voiding symptoms/dysfunctions (VS/Ds) after surgery for deep-infiltrating endometriosis (DIE) are frequent (20% of patients) and, together with bowel dysfunctions, may represent a de novo disorder due to surgical damage of the pelvic plexus or a worsening of pre-existent functional damage. Sacral neuromodulation (SNM) might improve voiding symptoms by treating dysfunctional voiding. The aim of this study is to report our experience with SNM in patients treated with surgery for DIE.We retrospectively enrolled 13 patients with VS/Ds after surgery for DIE. All patients were investigated with urodynamic studies (UDS) and agreed to undergo SNM. Pre-existing VS/Ds, bowel disorders and pelvic pain, DIE surgical procedures, UDS and SNM test results were recorded.After surgery for DIE, functional bladder outflow obstruction and detrusor acontractility were observed in nine and four patients, respectively. Chronic pelvic pain was present in seven cases. Twelve patients developed constipation, whilst one patient had de novo faecal incontinence. After the SNM testing period, nine patients (69.2%) experienced a significant improvement of symptoms that led to definitive implant. Four patients (30.8%) had no symptom relief and the system was removed.Functional bladder outflow obstruction and urinary retention are the most common VS/Ds after surgery for DIE. SNM may be an effective option for these patients, probably due to its action in improving the dysfunctional voiding, which was likely to be already present as part of the “endometriotic syndrome” and got worse after pelvic surgery. Results for pelvic pain control and gastrointestinal disorders should not be underestimated. Endometriosis is defined as the presence of functional endometrial glands and stroma outside the uterine cavity, predominantly, but not exclusively, in the pelvic compartment. It is an oestrogen-dependent chronic inflammatory condition that affects 5–10% of women in their reproductive life and is associated with pelvic pain and infertility [The term “deep-infiltrating endometriosis” (DIE) should be reserved for lesions in the retroperitoneal tissue  but, for practical purposes, it includes lesions penetrating anatomical pelvic structures, mainly of the posterior compartment (93.4% of cases) , such asUrinary symptoms  are present—depending on the methods and questionnaires used to highlight these conditions—in 5–20% of patients suffering from endometriosis, before any kind of surgery, but are often masked by the prevalence of gynaecological and gastrointestinal disorders, particularly in the deep-infiltrating forms .Following surgery for DIE, > 20% of patients experience voiding symptoms , mainly related to urinary retention and dysfunctional voiding. These voiding symptoms/dysfunctions (VS/Ds) may represent a de novo disorder related to surgical damage of the pelvic plexus or worsening of pre-existent functional damage –8.A few therapeutic alternatives for patients with persistent VS/Ds after surgery for DIE are available. Intermittent self-catheterisation remains the gold standard to treat urinary retention, to avoid recurrent urinary tract infections and urogenital prolapse due to abdominal straining. Dysfunctional voiding, which is related to poor relaxation of the external urethral sphincter (EUS), is thought to cause reflex inhibition of detrusor contraction by potentiating a spinal pro-continence reflex. The use of muscle-relaxant drugs to inhibit urethral sphincter activity has not been validated , and fewSacral neuromodulation (SNM), consisting of continuous electrical stimulation of the sacral segmental unilateral nerve S3, enables bladder emptying by peripheral (inhibition of guarding reflex) and central action . SNM hasHowever, few case series and validated data on SNM in the treatment of VS/Ds after surgery for DIE are available.The aim of this preliminary study is to report our experience with SNM in patients suffering from VS/Ds who came to urologist’s attention for the first time after surgery for DIE.We retrospectively enrolled 13 patients who underwent a SNM implant from April 2015 to March 2019 for persistent VS/Ds after pelvic surgery for deep-infiltrating endometriosis. All patients underwent invasive urodynamic studies (UDS) supplemented with needle electromyography of the external urethral sphincter (EUS EMG) before SNM.The study was conducted according to the Declaration of Helsinki general principles for medical research involving humans. Following our Institutional Ethical Committee Approval (protocol no. 0036753) and acquisition of informed consent, patient data were obtained  and collected in the Sacral Neuromodulation Database of our institution. We used SPSS software to analyse data and perform our computations.2O) in the presence of incomplete bladder emptying (post-voided volume > 100 ml and voided volume > 200 ml); an increase in EUS EMG activity during micturition [According to the International Continence Society (ICS) recommendations , a bladdturition . Bladderturition .Only three patients were investigated before the SNM test for pudendal neuropathy using bilateral pudendal nerve terminal motor latency (PNTML) and external anal sphincter electromyography (EAS EMG) studies. In PNTML evaluation, the delay between the electrical stimulation of the pudendal nerve and the EMG activity of the external anal sphincter (EAS) was recorded using a St. Mark’s glove electrode©. During EAS EMG, EMG activity was recorded by an EMG needle inserted in the four quadrants of the EAS.The SNM Interstim© staged procedure was performed using a standardised electrode placement technique . In the VS/Ds before SNM therapy, previous DIE surgical procedures, UDS results, time between surgery and SNM test, age at the SNM test, SNM test results and daily number of self-catheterisations (if performed) before the SNM test and after permanent implantation of the system were recorded for each patient.Patient characteristics are reported in Table Surgery for deep-infiltrating endometriosis involved the uterus, ovaries, utero-sacral ligaments, parametrium, recto-vaginal septum, Douglas pouch and peritoneum in most cases. Bilateral adnexectomy was performed in one case, and partial intestinal resection was performed in six cases.The main voiding dysfunction after surgery, assessed by UDS and EUS EMG and observed in nine patients (69.2%), was dysfunctional voiding due to overactive urethral function, defined as described above. Three out of nine patients with an outflow obstruction needed self-catheterisation (4 catheters per day) to complete bladder emptying. Main symptoms in this cohort of patients were dysuria, hesitancy, intermittent flow and incomplete bladder-emptying sensation. Bladder acontractility was observed in the remaining four patients (30.8%), for whom self-catheterisation was mandatory (4–5 catheters per day).Half of the patients with bladder acontractility underwent an intestinal resection during surgery for DIE.Intestinal constipation was present in 12 cases (92.3%), with use of trans-anal irrigation in only one patient. Faecal incontinence after surgery was observed in one case (7.7%).Pelvic pain was present in seven patients (53.8%) after surgery (mean VAS score: 7.5).Bilateral pudendal neuropathy was recorded in two out of three patients who underwent neurophysiological studies.After the SNM testing period, nine patients (69.2%) experienced a significant improvement of symptoms that led to definitive implant. As shown in Table Four patients (30.8%) had no symptom relief and the quadripolar tined-electrode was removed, including one of the two patients with the previously identified pudendal neuropathy.Pelvic surgery for deep-infiltrating endometriosis is frequently associated with VS/Ds, mainly because of incomplete bladder emptying and increased post-voided residual.On one hand, these symptoms could be justified by the aggressive surgical approach often adopted and the partial or total lesion of the inferior hypogastric and pelvic plexus, which lead to a hypo-acontractile bladder, with subsequent episodes of partial or total urinary retention. Ballester et al. showed that the rate of significant post-voiding residual volume after colorectal resection for endometriosis ranges from 15 to 20%. This rate seems to be higher (up to 30%) after proximal utero-sacral ligament resection. This might be explained by the location of the inferior hypogastric plexus at the proximal portion of the utero-sacral ligaments . MoreoveOn the other hand, voiding dysfunctions can be found in 5–20% of patient affected by endometriosis before any kind of pelvic surgery, and only a small number of these cases can be “anatomically” explained by localisation of endometriotic foci in the lower urinary tract , 22. A fAfter pelvic surgery, urinary symptoms are necessarily due to a combination of anatomical damage and pre-existing functional damage, which is part of the natural history of the disease.Whilst the organic lesions of the hypogastric and pelvic plexus cannot be restored yet, it is conceivable that SNM therapy, which has been successfully used for both treatment of unsustained bladder contraction and dysfunctional voiding , may leaSimilarly, only a few gastrointestinal disorders can be explained by the presence of endometriotic rectal nodules, and cyclic inflammatory phenomena that lead to irritation of bowel mucosa have been proposed as a contributory cause of bowel dysfunctions, such as constipation, in patients suffering from endometriosis prior to surgery. Moreover, pre-existent gastrointestinal symptoms can be associated with de novo bowel disorders because of radical surgery and pelvic denervation . ConsideA few case series about SNM in patients affected by endometriosis and treated with surgery are available.Lavonius et al. described their experience with SNM on pain-related symptoms in four women previously treated with pelvic surgery for DIE. Three patients obtained a benefit from SNM, intended as a reduction of pain drugs assumption or a significant improvement in validated questionnaire score .Nyangoh and colleagues retrospectively enrolled five patients with bladder acontractility and need for self-catheterisation due to pelvic surgery for DIE. After SNM and a 4-year follow-up, one patient stopped self-catheterisation because of a complete resumption of spontaneous micturition; a second patient reduced the daily catheters from 4 to 2, with post-voiding volume always < 100 ml .In our series, we retrospectively analysed 13 patients referred to our centre in a 47-month time period, for persistent VS/Ds after surgery for DIE. All patients underwent a standardised SNM implant procedure, performed by a single surgical team. Nine patients obtained a clinical benefit on bladder sensibility and emptying, pain control or bowel management during the testing phase and underwent definitive implant.All of our patients were treated with SNM after at least 2 years from the last pelvic surgery. According to different authors, spontaneous improvement of bladder function after pelvic surgery is possible up to 1–3 years after the surgery. Hence, it would appear logical to propose a SNM test for patients with VS/Ds lasting > 1 year . FurtherConsidering the possible serious damage of pelvic surgery on neural pathways, it might be helpful to include neurophysiological studies as part of the diagnostic evaluation of patients before offering SNM as a potential therapeutic option. Furthermore, a recent study shows how a peripheral neuropathy is already present in > 30% of patients before surgery due to endometriosis inflammation and nerve involvement . On the The small sample size, a lack of standardised diagnostic criteria and the retrospective nature of the study are limits that must be overcome to obtain strong data and predictive factors of SNM response in patients affected by endometriosis.VS/Ds and UDS findings after surgery for deep-infiltrating endometriosis should be explained by both anatomical damage of the inferior hypogastric/pelvic plexus and worsening of pre-existent functional damage which is part of the natural history of the disease.On one hand, better topographic understanding of the autonomic pelvic network should help to prevent iatrogenic injury through the adoption of nerve-sparing surgical techniques to reduce postoperative autonomic dysfunction. On the other hand, the use of SNM to improve the voiding dysfunction and functional damage is certainly worth considering. Results on pelvic pain control and gastrointestinal disorders should not be underestimated. The results of our study are encouraging. Further studies with greater number of patients and standardised diagnostic evaluations are required to assess the efficacy of SNM for the control of VS/Ds after surgery for DIE.


Postpartum depression, a subtopic of Behavioral Health: Abnormal Processes: Mood Disorders in the integrated United States Medical Licensing Examination (USMLE) Content Outline , presentWe  created a 110-min flipped classroom module on postpartum depression for the pre-clerkship psychiatry course in the 2nd-year medical student curriculum at our institution for the 2020–2021 academic year. Although originally planned as an in-person session, our school switched to online remote instruction due to the COVID-19 pandemic 2 months prior to its scheduled date. Due to this transition, we chose to make this session non-mandatory.We adapted our pre-clerkship postpartum depression flipped classroom case from a peer-reviewed OBGYN clerkship postpartum depression team-based learning session published by Freerksen et al. in 2015 . Our stuWe assessed students’ perceptions after the flipped classroom module via a voluntary, anonymous online survey, which was exempted by our Institutional Review Board. Completion of the survey implied consent to study participation. We sent an invitation to all students enrolled in the course to participate in the survey with reminders until 2 weeks following the session.Of the 123 second-year medical students at the University of California, Davis School of Medicine, 116 (94.3%) attended this non-mandatory flipped classroom module. We recorded student attendance over the course of the session using the Zoom webinar attendee report: 116 students participated in the readiness self-assessment quiz review, of which 97 participated in the first breakout session, 84 in the following large group discussion, 68 in the next breakout session, and 67 in the final large group discussion. At the beginning of the session, some students expressed confusion about whether the session was mandatory, which we clarified was not. Of the students who attended the session, 78% (91/116) responded to our survey. Most students felt that the preparation materials were helpful , that the pre-class quiz helped them assess their preparation for the session , and that the session met its learning objectives . Eighty-nine percent of students (80/90) agreed or strongly agreed that having instructors from both OBGYN and psychiatry in the session improved their understanding of postpartum depression, and most students found the peer discussion in the breakout rooms helpful for their learning . Students agreed or strongly agreed that the video visits helped them understand how to interview patients with postpartum depression  and how clinicians use telemedicine in patient care . Students largely felt that this session should be included in next year’s course .Our survey included two open-ended questions asking students what they found most useful about the flipped classroom module and how the session could be improved for future years. Over half of the respondents cited the video visits as most useful , followed by having instructors from both OBGYN and psychiatry present . Students also appreciated learning about postpartum depression , working in breakout groups , and preparing for the session via the review article  and the readiness self-assessment quiz . Several students  felt the breakout rooms could be improved, with most of these students suggesting that a facilitator be assigned to each breakout room. Some students also recommended changes to the self-assessment quiz  or cited technical difficulties . Three students requested more clarity about the optional nature of the session (9%), and three students wished that the psychiatry course’s lecture on depression had been available to view beforehand (9%). Finally, students advocated for using more gender-inclusive language  and for more case examples, real patient cases, or live patients .The COVID-19 pandemic has significantly impacted medical school curricula, requiring educators to deliver pre-clerkship coursework virtually through various methods –6. We usEfforts to teach about postpartum depression have focused on training medical students during their clinical clerkships in psychiatry  or OBGYNThe use of Zoom highlighted the differences between remote breakout rooms and traditional in-person flipped classroom sessions, the latter requiring only a few facilitators who can roam between student groups as needed. In the Zoom format, students desired more facilitator time in the breakout rooms to increase student engagement, which requires more facilitators. Although Zoom attendance showed attrition over the course of the session, we do not feel that attendance should be required during a pandemic. Non-mandatory, Zoom-based coursework can be more engaging than in-person coursework and may promote student comfort and autonomy during this stressful time . Other iDramatic changes in healthcare delivery have resulted from the COVID-19 pandemic. Specifically, physicians’ use of telemedicine and team-based care is greatly expanding, and medical students need to be prepared earlier to develop the skills necessary to deliver high-quality collaborative care online . Our fli


Cortical mean diffusivity is a novel imaging metric sensitive to early changes in neurodegenerative syndromes. Higher cortical mean diffusivity values reflect microstructural disorganization and have been proposed as a sensitive biomarker that might antedate macroscopic cortical changes. We aimed to test the hypothesis that cortical mean diffusivity is more sensitive than cortical thickness to detect cortical changes in primary progressive aphasia (PPA).GRN-related PPA) as well as 89 controls from three centers. The 3-Tesla MRI protocol included structural and diffusion-weighted sequences. Disease severity was assessed with the Clinical Dementia Rating scale. Cortical thickness and cortical mean diffusivity were computed using a surface-based approach.In this multicenter, case-control study, we recruited 120 patients with PPA (52 non-fluent, 31 semantic, and 32 logopenic variants; and 5 The comparison between each PPA variant and controls revealed cortical mean diffusivity increases and cortical thinning in overlapping regions, reflecting the canonical loci of neurodegeneration of each variant. Importantly, cortical mean diffusivity increases also expanded to other PPA-related areas and correlated with disease severity in all PPA groups. Cortical mean diffusivity was also increased in patients with very mild PPA when only minimal cortical thinning was observed and showed a good correlation with measures of disease severity.Cortical mean diffusivity shows promise as a sensitive biomarker for the study of the neurodegeneration-related microstructural changes in PPA.The online version contains supplementary material available at 10.1186/s13195-022-00974-0. Primary progressive aphasia (PPA) encompasses different neurodegenerative syndromes characterized by prominent deterioration of speech and language, and relative sparing of other cognitive functions , 2. In 2Currently, there are no disease-modifying treatments for PPA, but many pharmacological and non-pharmacological treatments are being developed with promising results . HoweverQuantitative analysis of brain structure with magnetic resonance imaging (MRI) can detect disease-specific abnormalities and enable the comparison with normal populations and the determination of the vulnerable regions in each syndrome. Previous MRI studies have shown that each PPA variant is characterized by a relatively focal onset, and spreading along highly connected cerebral regions –12. To dIn this multicenter study, our objective was to compare the cortical thickness and cortical mean diffusivity changes in patients with PPA. We also aimed to correlate these changes with clinical measures of disease severity. We hypothesized that cortical mean diffusivity is more sensitive than cortical thickness to detect cortical changes in PPA.Participants with PPA were recruited from two cohorts, at three different centers: 88 at the Memory and Aging Center (MAC) of University of California, San Francisco , and 72 at the Catalan Frontotemporal Dementia Initiative . In both cohorts, the diagnosis was made by neurologists with expertise in the evaluation of PPA , 20. AllGRN gene (n = 5) were classified in a separate group because these cases are characterized by a widespread and asymmetric pattern of gray matter loss  and with 18F-Florbetapir at CATFI [n =4]). The core AD biomarkers were available for CATFI participants and were performed as previously described [Amyloid PET and core AD biomarkers were also available in a subset of participants. Amyloid PET studies were read as positive or negative, as previously described , and werescribed , 31.Neuropathologic assessments performed at UCSF or Barcelona’s Neurological Tissue Bank followed previously described procedures , 21. ParAll participants underwent a 3T MRI using 4 different scanners. The acquisition parameters by the scanner can be found in Supplementary Table Cortical thickness reconstruction was performed with the Freesurfer package v6  using a previously described procedure . All indb = 0 image and the diffusion-weighted acquisitions. Then, after removing non-brain tissue using the Brain Extraction Tool, diffusion tensors were fitted, and mean diffusivity was computed using the FSL’s dtifit command. We then computed the affine transformation between the skull-stripped b0 and the segmented T1-weighted volume using a boundary-based algorithm as implemented in Freesurfer’s bbregister. This approach takes advantage of the accurate segmentation of the white matter surface and pial surface obtained during Freesurfer’s segmentation , to accurately register the b0 and the T1-weighted image, maximizing the intensity gradient across gray matter and white matter between both volumes. At this point, all the diffusion to T1 registrations were visually inspected to exclude those subjects with an erroneous co-registration. Seven participants were excluded due to diffusion-weighted imaging processing errors. Then, the mean diffusivity volume for each individual was sampled at the midpoint of the cortical ribbon  and projected to each surface reconstruction obtained during the Freesurfer processing, to create a surface map of cortical mean diffusivity (using Freesurfer’s mri_vol2surf command). Finally, individual cortical mean diffusivity maps were normalized to an average standard surface using a spherical registration, enabling an accurate inter-subject matching of cortical locations for the statistical analyses. Before statistical analyses, we applied a Gaussian kernel of 15 mm as implemented in Freesurfer [We used a previously described home-made surface-based approach to process cortical diffusion MRI . Recent eesurfer , to obtaBecause diffusion tensor imaging metrics are very sensitive to acquisition parameters, harmonization approaches are required to mitigate center-specific differences in multicenter studies. We applied a multicenter harmonization algorithm based on ComBat, to reduce center-specific differences in cortical mean diffusivity quantifications before any statistical analysis . Brieflyt-test for continuous variables (with Bonferroni correction for multiple comparisons) and the chi-square for dichotomous or categorical data. We first performed group comparisons for cortical thickness and cortical mean diffusivity with a two-class general linear model, as implemented in Freesurfer, comparing each PPA variant to the control group. These analyses were repeated for each center independently. Moreover, we also performed additional analyses including only PPA participants with a global CDR® plus NACC FTLD score of 0.5 (mild PPA). Next, we performed a vertex-wise correlation analysis in each PPA variant group between the cortical thickness and cortical mean diffusivity and the CDR® plus NACC FTLD-SB  and the MMSE . Specifically, a general linear model was created in which cortical mean diffusivity or cortical thickness was included as the dependent variable, and CDR® plus NACC FTLD-SB scores (and MMSE scores) were independent variables. We included age, sex, handedness, and center as nuisance variables in the cortical thickness analysis. In mean diffusivity analysis, only age, sex, and handedness were included since diffusion tensor imaging data were already harmonized between centers in a previous step. Only results that survived multiple comparisons (family-wise error < 0.05) based on Monte Carlo simulation with 10,000 repeats as implemented in Freesurfer are presented. We used a very stringent threshold of α = 0.001 for the group analyses including all PPA participants and HC, and a threshold of α = 0.05 for the analyses including PPA subgroups and the correlation analyses. A full description of the multiple comparison’s methodology can be found in the Data were explored for normality using the Shapiro-Wilk test. When necessary, variables were log-transformed using the natural log to fulfill the normal distribution assumption. Between-group differences were determined with ANOVA or d effect size metric for both cortical thickness and cortical mean diffusivity, on a vertex-wise basis, to obtain a topographical representation of the effect size for the group comparison between participants with PPA and controls. Effect size computation was restricted to cortical regions showing statistically significant differences between participants with PPA and controls for either cortical thickness or cortical mean diffusivity. We then computed the difference between the cortical thickness and cortical mean diffusivity effect size maps to obtain a topographical representation of the net effect size for each metric.We computed the Cohen’s GRN groups. APOEɛ4 carriers were overrepresented in the lvPPA group (33%) compared to the nfvPPA, svPPA, and GRN-PPA groups . The CDR® plus NACC FTLD-SB scores were higher in the svPPA group when compared to the nfvPPA group. The frequency of PPA participants with a global CDR® plus NACC FTLD score of 0.5 ranged from 19% in svPPA to 38% in nfvPPA.Table n = 8) had a diagnosis of lvPPA. Conversely, all participants with a diagnosis of FTLD were classified in the nfvPPA (n = 16), svPPA (n = 3), and GRN-PPA (n = 2) groups.As shown in Table We first compared cortical thickness and cortical mean diffusivity between PPA and controls. As shown in Fig. Thus, while cortical thickness and cortical mean diffusivity maps showed a partial overlap in all PPA variants, cortical mean diffusivity changes with at least moderate effect sizes extended beyond the areas of cortical thinning Fig. . ImportaGRN mutation and controls. As expected, we observed cortical thinning in the prefrontal, lateral temporal, and inferior parietal cortex. In this group, cortical thinning was restricted to the left hemisphere. In the PPA-GRN group, we also observed widespread increases in cortical mean diffusivity affecting both hemispheres . As expected, we observed the same pattern of cortical thinning and cortical mean diffusivity increases. However, these changes were more restricted to the neurodegeneration cores of each PPA variant (Fig. Finally, we evaluated the capacity of cortical thickness and cortical mean diffusivity to reflect disease severity in PPA as measured by the CDR® plus NACC FTLD-SB scores. As shown in Fig. This large multicenter study investigated cortical mean diffusivity in PPA. Importantly, we showed that cortical mean diffusivity increases not only coincided with areas that showed cortical thinning but also involved other areas that typically become affected later during disease progression. Cortical mean diffusivity was increased in patients with very mild PPA when only minimal cortical thinning was observed. Finally, we also explored the correlation between cortical mean diffusivity and clinical measures of disease severity and general cognitive function. Taken together, these findings suggest that cortical mean diffusivity might be more sensitive than cortical thickness to detect the earliest disease-related cortical changes in PPA.Cortical mean diffusivity has been recently proposed as a sensitive biomarker for the detection of the earliest cortical changes in sporadic Alzheimer’s disease and the Amyotrophic Lateral Sclerosis-Frontotemporal dementia continuum , 16–18. PPA variants are characterized by a focal onset of neurodegeneration spreading along specific networks . We obseGRN mutations. We classified PPA participants with GRN mutations in a separate group because these cases are characterized by a more widespread and asymmetric pattern of gray matter loss. In the PPA-GRN group, cortical thinning was restricted to the left hemisphere, but cortical mean diffusivity extended to both hemispheres. This finding also supports the view that cortical mean diffusivity is capturing early neurodegenerative changes antedating overt neuronal loss and cortical thinning. This observation in the PPA-GRN group also encourages the investigation of cortical mean diffusivity in larger samples of FTLD mutation carriers.Furthermore, we performed an exploratory analysis in the PPA participants with The suggestion that cortical mean diffusivity may be more sensitive than cortical thickness to detect the PPA cortical changes is further supported by our correlation analyses with the CDR® plus NACC FTLD-SB and MMSE scores. The CDR® plus NACC FTLD-SB has been validated as a tool for disease monitoring in clinical trials , 41. AltGRN mutation, the nfvPPA syndrome is frequently associated with Tau subtypes of FTLD, while the svPPA is associated with the TDP-C subtype of FTLD, and lvPPA is typically associated with AD pathologic changes [Although clinical-pathological correlations are not perfect, each PPA variant is associated with typical pathologies. For instance, in the absence of a  changes . We obse changes . Importa changes . Second, changes . Because changes . However changes –48.Our findings support the role of cortical mean diffusivity as a potential neurodegeneration biomarker in PPA to be used in clinical trials. Trials of drugs targeting abnormal protein deposition need meaningful end points. Historically, these end points included neuropsychological scores and functional scales. Brain imaging could provide more sensitive and robust evidence of disease modification . Our stuThe main strengths of this study are the relatively large number of participants with PPA at a mild disease stage, and the surface-based analyses using a previously validated technique. This surface-based approach solves some of the limitations and methodological concerns that have been previously reported when using a voxel-based approach . MoreoveGRN group and the subgroup of PPA participants at a mild disease stage were small. Finally, further longitudinal studies in presymptomatic mutation carriers should confirm that cortical mean diffusivity changes track disease progression and antedate cortical atrophy in patients with PPA.This study has also some limitations. A proportion of participants with PPA were excluded due to segmentation or diffusion tensor imaging processing errors. Even though this is an inherent limitation of our surface-based approach, future improvements in T1 MRI acquisitions or the use of higher field MRIs will likely reduce the number of subjects excluded due to segmentation errors. Also, both the PPA-Cortical mean diffusivity shows promise as a sensitive biomarker for the study of the earliest neurodegeneration-related cortical changes in PPA. Further longitudinal studies including preclinical mutation carriers are needed to fully determine the potential utility of this biomarker to detect cortical changes antedating cerebral atrophy at the earliest stages of the disease and to track disease progression.Additional file 1. Supplementary material.


Pelvic exenteration is a complex procedure performed for the management of advanced pelvic cancers. It often involves the resection of several pelvic organs and can be associated with a high morbidity and impact on the patient’s quality of life. The development of better diagnostics, improved chemotherapy and radiotherapy regimens, combined with advanced surgical strategies have improved surgical and survival outcomes. This article highlights current and future management options.Pelvic exenteration is a complex operation performed for locally advanced and recurrent pelvic cancers. The goal of surgery is to achieve clear margins, therefore identifying adjacent or involved organs, bone, muscle, nerves and/or vascular structures that may need resection. While these extensive resections are potentially curative, they can be associated with substantial morbidity. Recently, there has been a move to centralize care to specialized units, as this facilitates better multidisciplinary care input. Advancements in pelvic oncology and surgical innovation have redefined the boundaries of pelvic exenterative surgery. Combined with improved neoadjuvant therapies, advances in diagnostics, and better reconstructive techniques have provided quicker recovery and better quality of life outcomes, with improved survival This article provides highlights of the current management of advanced pelvic cancers in terms of surgical strategy and potential future developments. Pelvic exenteration is a complex operation performed for locally advanced and recurrent pelvic cancers . The proThe COVID-19 pandemic has disrupted patient access to healthcare worldwide, despite efforts to maintain essential cancer care during this time ,11. ThisAdvancements in pelvic oncology and surgical innovation have redefined the boundaries of pelvic operations . AggressPrecise tumour staging is essential to determine prognosis and enables a more focused assessment of the available management options . Local sThe use of MRI of the chest/thorax is not widely available and is subject to significant operator variation . HoweverRegardless of modality, good communication and feedback between surgical and radiological departments is critical in the assessment of patient operability and for predicting resectability. Expertise is required to devise a radiological roadmap that incorporates surgical planes, highlighting potential issues and ensuring negative margins. A roadmap for R0 excision should be tailored to the maximum possible disease extent previously identified on sequential MRI imaging, regardless of down-staging post-neoadjuvant treatment. The rationale behind this is based on the knowledge that radiologically occult microscopic foci of viable tumour cells can occasionally persist beyond down-staged tumour margins . For this reason, areas of fibrosis in contact with tumours on post-treatment imaging should be considered to have malignant potential and included in the extended resection ,28,29,30Total neoadjuvant therapy (TNT) is increasingly utilized for the downstaging of locally advanced rectal cancer (LARC) . Recent The treatment response to neoadjuvant chemoradiotherapy is highly variable, with up to 20% of rectal tumours exhibiting a complete resistance . The actProgrammed cell death-1 (PD-1) is a cell receptor found on the surface of activated T-cells, pro-B cells and macrophages that contains at least two ligands; programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2). The binding of these two ligands to the PD-1 receptor results in T-cell deactivation and subsequent tumour cell evasion, preventing a host attack on its own immune system . PD pathPerioperative strategies to optimise the outcomes of patients undergoing pelvic exenteration or extended resection for pelvic cancers is critical to maximising treatment success . Pre-exiSurgical patients with a poor functional capacity, determined by oxygen consumption at anaerobic threshold (AT) during cardiopulmonary exercise testing (CPET), experience poorer post-operative outcomes. The identification of high-risk surgical patients allows for the appropriate planning of their perioperative care, subsequently reducing the risk of mortality or severe complications in the post-operative period . Pre-opeWhere possible, the anaesthetist undertaking the case should personally pre-assess the high-risk patient undergoing exenteration;When available, CPET should be utilised to assess functional capacity pre-operatively;Pelvic exenteration can be undertaken in patients who have demonstrated an adequate CPET result and have been deemed low risk for severe perioperative morbidity;Patients with more than two cardiac risk factors and poor functional capacity should undergo imaging stress-testing prior to surgery;Formal cardiology assessment is not routinely required in patients undergoing pelvic exenteration.Cancer-related malnutrition occurs secondary to anorexia, nausea, vomiting, metabolic disorders and psychological factors in patients undergoing major oncological surgery. Two cohort studies identified that 32.5% and 24% of their populations, respectively, were malnourished before exenteration when assessed by the subjective global assessment (SGA) tool . The optNegative resection margins (R0) are the single most important prognostic factor in predicting long-term survival in patients undergoing pelvic exenteration ,53,54,55Historically, the presence of hydronephrosis, gross lower limb oedema, and invasion of the sciatic notch or involvement of the aortoiliac axis suggested inoperable disease. Various studies have demonstrated the safety and efficacy of major extra-anatomic resections involving these structures in selective patients ,65,66. EWhile the extent of resection is theoretically limitless, it is imperative that morbidity is minimized and, therefore, operations are tailored to each patient. As advancements in reconstructive methods and rehabilitative systems are made, the indications and contraindications for surgery are constantly changing . CurrentIORT delivers a single high-fraction dose (10–20 Gy) of radiation directly to anatomical targets deemed as having the potential for high recurrence risk . IORT isIORT can be delivered via two methods: high-dose-rate brachytherapy or intraoperative electron beam radiotherapy (IOERT). The evidence for IORT stems from positive prospective long-term data from several international units who administer it when there is concern for threatened margins . A recenCurrent treatment options for patients with peritoneal metastases include supportive care, palliative systemic chemotherapy, pressurised intraperitoneal aerosol chemotherapy (PIPAC), and cytoreductive surgery (CRS) combined with heated intraperitoneal chemotherapy (CRS-HIPEC). When treated with modern systemic chemotherapy, these patients have a median survival of thirteen months . PatientCRS-HIPEC is a well-established treatment modality for patients with synchronous or metachronous colorectal peritoneal metastases . While iFurther research is required via a multi-centre approach to determine the optimal candidates for this approach . The PROFor patients presenting with rectal cancer, 15–20% will have synchronous liver metastases . The optThe PelvEx Collaborative observed an R0 resection in 73.5% of pelvic exenterations and 66.4% of liver resections among 128 patients with synchronous liver metastases. The 5-year overall survival for patients in whom an R0 resection was achieved was 54.6% in comparison to 20% for those with an R1/R2 resection. This was the first multi-centre study that demonstrated the safety and feasibility of simultaneous liver resection, with acceptable morbidity and mortality rate .Debate around the optimal treatment of colorectal lung metastases also remains. . The majMinimally invasive surgical modalities have evolved considerably in recent years, particularly regarding pelvic procedures . A recenIn the PelvEx Collaborative meta-analysis comparing MIS techniques to the open approach, 78.1% underwent open exenteration while 21.8% had an MIS exenteration analysis among 170 patients. MIS exenteration was associated with a longer operating time but substantially less blood loss. MIS exenteration was also associated with a significantly reduced overall morbidity rate (56.7% versus 88.5%) and a short post-operative length of stay (6 days less). This study demonstrated the safety and feasibility of MIS exenteration in patients with favourable anatomy and tumour characteristics . FluoresRadical resections incur a greater need for reconstruction. The ability to perform complex soft tissue, vascular and bone reconstruction/stabilization has improved the functional outcomes of patients undergoing pelvic exenteration.Abdominal: Vertical or oblique rectus abdominus myocutaneous (VRAM/ORAM).Gluteal: Myocutaneous or fascio-cutaneous VY-plasty, inferior gluteal artery perforator (IGAP) flap.Upper thigh: Anterolateral thigh +/− vastus lateralis flap, bilateral pedicled gracilis flap.Gluteal fold/perineal: Internal pudendal artery perforator or perineal turnover perforator flap.Many patients will require flap reconstruction after exenterative surgery due to extensive tissue loss. However, some co-existing factors will determine the feasibility of each specific reconstruction. Previous chemoradiotherapy, increased pelvic dead space, poor tissue vascular supply, accumulation of fluid and bacterial contamination all play a role in the development of flap complications, which occur in 25–60% of reconstructions . The folVaginal defects resulting from radical oncologic resection are challenging to reconstruct. These defects may range from simple mucosal defects to full circumferential loss due to posterior vaginal wall resection. Anatomy and function can be restored using a rectus abdominis myofascial flap, deep inferior epigastric perforator (DIEP) flap, bilateral gracilis flats or gluteus maximus special flaps .Empty pelvis syndrome is a major contributor to morbidity following pelvic exenteration as dead space allows for the accumulation of fluid and small bowel migration (obstruction) into the pelvis. To alleviate the risk of these complications, the dead space must be “filled” using either synthetic mesh or tissue. Reconstructive methods include myocutaneous flap reconstruction, omental flaps and mesh reconstruction. There is currently insufficient evidence in the literature to support the use of one reconstructive method over another .En bloc sacrectomy is performed in cases where tumours infiltrate the presacral fascia and may require further reconstruction . SacrectThe involvement of aortoiliac vessels, the sciatic nerve or its associated roots substantially increases the difficulty of achieving an R0 resection in advanced pelvic malignancies . To achiPalliative procedures, such as a defunctioning stoma or urinary diversion, can be performed in patients who are suffering from disabling symptoms where an R0 resection is unlikely . The decA recent meta-analysis performed by the PelvEx Collaborative demonstrated symptom relief in ‘select patients’ undergoing palliative exenteration. Symptom relief was reported in a median of 79% of patients, although the magnitude of the effect was poorly measured. Though available data are limited (23 studies comprising 509 patients), the results suggest that there may be some improvement in symptom control in selective patients. However, palliative exenteration is an extremely morbid procedure with insufficient evidence of sustained quality of life . As the Enhanced recovery after surgery (ERAS) protocols are well-established and have demonstrated improvements in morbidity rates, length of stay and quality of life . There iHarji et al. enrolled 145 patients into a dedicated pelvic exenteration ERAS programme to assess its feasibility and short-term impact on this cohort of patients. They demonstrated an overall compliance rate of 70%. Patients with higher compliance to the program tended to have a shorter hospital length of stay, reduced rate and severity of post-operative morbidity, as well as fewer readmissions. ERAS appears feasible and efficacious in patients undergoing pelvic exenteration, displaying a high compliance and improved clinical outcomes . See FigThe management of advanced colorectal malignancies continues to evolve rapidly, with the introduction of new technologies, pushing the boundaries of surgical resection and involvement in clinical trials and collaborative research. Increasingly, the focus of treatment is balanced between cure and quality of life . The IMPRadiomics and radiogenomics are being investigated as a novel way to analyse images and to increase the precision of diagnostics . The incThe radiomics analysis of contrast-enhanced CT images has already demonstrated improved accuracy for nodal assessment in advanced rectal cancer . SimilarRadiogenomics is the extension of radiomics by its combination with molecular analysis in the form of genomic and transcriptomic data. Genetic analysis remains expensive, invasive, and time-consuming. Radiogenomics may play a vital role in providing imaging surrogates that correlate with genetic expression, thereby providing an alternative to genetic testing . Of courThe adenoma-carcinoma sequence was first described in 1990 by Vogelstein and Fearon and provided the foundation for our understanding of colorectal cancer as a disease consisting of complex genomic changes . ComparaBRAF V600E is the most common potentially targetable mutation in metastatic colorectal cancer; however, RAF inhibitors have limited efficacy as single agents in treating patients with this alteration . The BEAHER2 amplification occurs in 2–6% of metastatic colorectal cancers and is associated with a poor response to EGFR antibody treatment . While aOur understanding of tumour biology and molecular subtypes is constantly expanding, thanks to advancements in microarray and NGS technologies which allow for the identification of new cancer genes and pathways. While translational genomic studies have already provided clinically relevant biomarkers for predicting prognosis and therapy response, future research will identify new drug targets and reveal novel therapeutic opportunities. Innovations in current applications, coupled with novel emerging technologies, will lead to further advancements in translational cancer genomics which will hopefully contribute to improved patient outcomes in the future .Over the last two decades, experienced exenterative surgeons have redefined what constitutes resectable disease. The development of regional/national specialized units has allowed funding and structural/service supports to enable these centres to establish specialist pelvic oncology units. As a result, extended bony and/or neurovascular resections are pursued more frequently, with an acceptable morbidity reported ,65,102.‘Higher and wider’ resections at the periphery of the pelvis are now commonplace in pelvic exenteration . CertainImproved reconstructive techniques have facilitated these more radical resections ,65,98,99Anatomical reconstruction of the sacrum using 3-dimensional printing technology has been sparsely reported in the literature ,130. KimCurrent options for tissue reconstruction rely heavily on autologous donor tissue to repair defects. 3D bioprinting offers the potential to avoid autologous grafts and the complications associated with them . Its useMagnetic resonance-guided histopathology has been shown to increase the accuracy of staging in LARC . The tecBiobanking is the process of collecting and storing various human specimens for the purpose of clinical research and provides a platform for the development of translational and personalised “precision” medicine . TranslaBiobanks provide researchers with human specimens and associated clinical data, which allow for large cohorts of over 30 specimens to be analysed using large-scale genome sequencing. This facilitates the discovery of novel molecular alterations as well as the classification of tumour subtypes according to distinct genomic alterations, providing a personalised, precision medicine approach in cancer care .Aggressive multi-visceral resections are performed more often to manage patients with advanced pelvic cancers . CurrentPatient counselling with shared decision-making is crucial in the consideration and planning of extensive pelvic surgery. A growing volume of research has shown that when patients are actively involved in decision-making and prehabilitation  that they experience better outcomes ,145. TheThe role of radical surgery in the setting of locally advanced and recurrent rectal cancer has evolved substantially. Novel strategies including TNT, cytoreductive and/or bony/vascular resection and enhanced reconstructive techniques have enabled surgeons to pursue what was once considered a terminal disease. Advancements in surgical technology, in particular the incorporation of artificial intelligence and three-dimensional bioprinting, will undoubtedly enhance our ability to move the limits of what is reasonable and possible to resect.