Understanding variants of unknown significance and classification of genomic alterations.

Despite recent efforts to issue clinical guidelines outlining strategies to define the pathogenicity of genomic variants, there is currently no standardized framework for which to make these assertions. This review does not present a step-by-step methodology, but rather takes a holistic approach to discuss many aspects which should be taken into consideration when determining variant pathogenicity. Categorization should be curated to reflect relevant findings within the scope of the specific medical context. Functional characterization should evaluate all available information, including results from literature reviews, different classes of genomic data repositories, and applicable computational predictive algorithms. This article further proposes a multidimensional view to infer pathogenic status from many genomic measurements across multiple axes. Notably, tumor suppressors and oncogenes exhibit fundamentally different biology which helps refine the importance of effects on splicing, mutation interactions, copy number thresholds, rearrangement annotations, germline status, and genome-wide signatures. Understanding these relevant datapoints with thoughtful perspective could aid in the reclassification of variants of unknown significance (VUS), which are ambiguously understood and currently have uncertain clinical implications. Ongoing assessments of VUS examining these relevant biological axes could lead to more accurate classification of variant pathogenicity interpretation in diagnostic oncology.

Structure and in vivo psoralen DNA crosslink repair activity of mycobacterial Nei2.

Mycobacterium smegmatis Nei2 is a monomeric enzyme with AP ß-lyase activity on single-stranded DNA. Expression of Nei2, and its operonic neighbor Lhr (a tetrameric 3'-to-5' helicase), is induced in mycobacteria exposed to DNA damaging agents. Here, we find that nei2 deletion sensitizes M. smegmatis to killing by DNA inter-strand crosslinker trimethylpsoralen but not to crosslinkers mitomycin C and cisplatin. By contrast, deletion of lhr sensitizes to killing by all three crosslinking agents. We report a 1.45 Å crystal structure of recombinant Nei2, which is composed of N and C terminal lobes flanking a central groove suitable for DNA binding. The C lobe includes a tetracysteine zinc complex. Mutational analysis identifies the N-terminal proline residue (Pro2 of the ORF) and Lys51, but not Glu3, as essential for AP lyase activity. We find that Nei2 has 5-hydroxyuracil glycosylase activity on single-stranded DNA that is effaced by alanine mutations of Glu3 and Lys51 but not Pro2. Testing complementation of psoralen sensitivity by expression of wild-type and mutant nei2 alleles in ∆nei2 cells established that AP lyase activity is neither sufficient nor essential for crosslink repair. By contrast, complementation of psoralen sensitivity of ∆lhr cells by mutant lhr alleles depended on Lhr's ATPase/helicase activities and its tetrameric quaternary structure. The lhr-nei2 operon comprises a unique bacterial system to rectify inter-strand crosslinks.IMPORTANCEThe DNA inter-strand crosslinking agents mitomycin C, cisplatin, and psoralen-UVA are used clinically for the treatment of cancers and skin diseases; they have been invaluable in elucidating the pathways of inter-strand crosslink repair in eukaryal systems. Whereas DNA crosslinkers are known to trigger a DNA damage response in bacteria, the roster of bacterial crosslink repair factors is incomplete and likely to vary among taxa. This study implicates the DNA damage-inducible mycobacterial lhr-nei2 gene operon in protecting Mycobacterium smegmatis from killing by inter-strand crosslinkers. Whereas interdicting the activity of the Lhr helicase sensitizes mycobacteria to mitomycin C, cisplatin, and psoralen-UVA, the Nei2 glycosylase functions uniquely in evasion of damage caused by psoralen-UVA.

Sit-to-Stand Power Is a Stronger Predictor of Gait Speed than Knee Extension Strength.

With a growing aging population, the routine assessment of physical function may become a critical component of clinical practice. The purpose of this cross-sectional study is to compare two common assessments of muscular function: (1) isometric knee extension strength (KES) and (2) sit-to-stand (STS) muscle power tests, in predicting objective physical function (i.e., gait speed) in aging adults. 84 adults (56% female, mean (SD) age = 66.6 (9.4) years) had their relative KES, STS power, usual gait speed (UGS), and fast gait speed (FGS) assessed. Multiple linear regression examined the associations between KES, STS power, and gait outcomes. When entered in separate models, KES and STS power were both independently associated with UGS and FGS (Std. ß = 0.35-0.44 and 0.42-0.55 for KES and STS power, respectively). When entered in the same model, STS power was associated with UGS and FGS (Std. ß = 0.37 [95%CI: 0.15, 0.58] and 0.51 [95%CI: 0.31, 0.70], respectively), while KES was only associated with FGS (Std. ß = 0.25 [95%CI: 0.02, 0.48]). STS power seems to be a valid indicator of function in aging adults. Its feasibility as a screening tool for "low" function in the primary care setting should be explored.

Watch and Wait in Rectal Cancer Patients with Residual Mucin on MRI following Neoadjuvant Therapy.

BACKGROUND: Neoadjuvant therapy (NAT) leads to a clinical complete response (cCR) in a significant proportion of patients with locally advanced rectal cancer (LARC), allowing for possible nonoperative management. The presence of mucin on MRI after NAT leads to uncertainty about residual disease and appropriateness of a watch-and-wait (WW) strategy in patients with no evidence of disease on proctoscopy (endoscopic cCR). METHODS: MRI reports for LARC patients seen between July 2016 and January 2020 at Memorial Sloan Kettering Cancer Center were queried for presence of mucin in the tumor bed on MRI following NAT. Clinicodemographic, pathologic, and outcome data were compiled and analyzed. RESULTS: Of 71 patients with mucin on post-treatment MRI, 20 had a cCR and 51 had abnormalities on endoscopy and/or physical exam. One patient with a cCR opted out of WW; thus, 19 patients (27%) entered WW and 52 patients (73%) were planned for surgery (Non-WW). Of the 19 WW patients, 15 (79%) have had no local regrowth with median follow-up of 50 months (range, 29-76 months), while 4 (21%) experienced regrowth between 9 and 29 months after neoadjuvant therapy. Of the 52 patients who were planned to have surgery (Non-WW), 49 underwent resection while 3 developed metastatic disease that precluded curative-intent surgery. Five (10%) of the 49 patients who underwent surgery, including the one with an endoscopic cCR, had a pathologic complete response. CONCLUSIONS: The presence of mucin after NAT for LARC does not preclude WW management in otherwise appropriate candidates who achieve an endoscopic cCR.

Direct prediction of intermolecular interactions driven by disordered regions.

Intrinsically disordered regions (IDRs) are critical for a wide variety of cellular functions, many of which involve interactions with partner proteins. Molecular recognition is typically considered through the lens of sequence-specific binding events. However, a growing body of work has shown that IDRs often interact with partners in a manner that does not depend on the precise order of the amino acid order, instead driven by complementary chemical interactions leading to disordered bound-state complexes. Despite this emerging paradigm, we lack tools to describe, quantify, predict, and interpret these types of structurally heterogeneous interactions from the underlying amino acid sequences. Here, we repurpose the chemical physics developed originally for molecular simulations to develop an approach for predicting intermolecular interactions between IDRs and partner proteins. Our approach enables the direct prediction of phase diagrams, the identification of chemically-specific interaction hotspots on IDRs, and a route to develop and test mechanistic hypotheses regarding IDR function in the context of molecular recognition. We use our approach to examine a range of systems and questions to highlight its versatility and applicability.

Neuroblastoma Predisposition and Surveillance-An Update from the 2023 AACR Childhood Cancer Predisposition Workshop.

Genetic predisposition to neuroblastoma (NB) is relatively rare. Only 1% to 2% of patients have a family history of NB, 3% to 4% of cases present with bilateral or multifocal primary tumors, and occasional patients have syndromes that are associated with increased NB risk. Previously, a germline pathogenic variant (GPV) in PHOX2B was associated with Hirschsprung disease and congenital central hypoventilation syndrome. Recently, certain GPVs were shown to be responsible for congenital central hypoventilation syndrome and NB predisposition. Also, several groups determined that activating GPVs in ALK accounted for a substantial number of familial NB. Finally, there are additional genes and cancer predisposition syndromes in which NB occurs with greater frequency or that have been associated with NB based on genome-wide association studies. We review the evidence for all these genes and whether there is sufficient evidence to warrant surveillance. We review recommended surveillance for hereditary patients with NB, including minor updates to surveillance recommendations that were published previously in 2017.

Educational and Clinical Outcome Measures in an Integrated Hand Service.

Background: We recently sought to integrate our orthopaedic and plastic hand surgeons with the goal of improving education, patient care, and providing seamless, continuous coverage for our trauma center. Our hypothesis was that integration could serve both the orthopaedic and plastic surgery training programs well and provide more consistent care for the trauma patients. Materials and Methods: Program director approval was granted for blinded analysis of case logs from plastic and orthopaedic surgery programs from 2012 through 2019. Data on mean and total number of hand cases were analyzed and compared for both specialties. Institutional Review Board approval was granted for a retrospective review of patient outcomes. Results: For both orthopaedic and plastics resident trainees, the mean number of hand cases increased during this study period suggesting that the integration had a favorable impact on both programs. The mean number of hand cases for orthopaedic residents rose from 163 to 246. The mean number of hand cases for plastic surgery residents rose from 218 to 295. Patient outcomes as reflected in length of stay and time to consultation also improved. Conclusion: To improve hand surgical training and patient care, an integrated orthoplastics approach to hand surgery was implemented at our institution. Plastic surgery trainees are completing more hand surgery cases in an integrated model (p < 0.001), including fracture care (p < 0.047). Orthopaedic surgery trainees have doubled the percentage of integumentary and microsurgery cases in the integrated model (p < 0.001). The educational and clinical changes affected in an integrated model have changed the paradigm for educating future hand surgeons at our institution.

Identification of TP53 germline variants in pediatric patients undergoing tumor testing: strategy and prevalence.

BACKGROUND: TP53 alterations are common in certain pediatric cancers, making identification of putative germline variants through tumor genomic profiling crucial for patient management. METHODS: We analyzed TP53 alterations in 3123 tumors from 2788 pediatric patients sequenced using tumor-only or tumor-normal paired panels. Germline confirmatory testing was performed when indicated. Somatic and germline variants were classified following published guidelines. RESULTS: In 248 tumors from 222 patients, 284 Tier 1/2 TP53 sequence and small copy number variants were detected. Following germline classification, 73.9% of 142 unique variants were pathogenic/likely pathogenic (P/LP). Confirmatory testing on 118 patients revealed germline TP53 variants in 28 patients (23 P/LP and 5 uncertain significance), suggesting a minimum Li-Fraumeni syndrome (LFS) incidence of 0.8% (23/2788) in this cohort, 10.4% (23/222) in patients with TP53 variant-carrying tumors, and 19.5% (23/118) with available normal samples. About 25% (7/28) of patients with germline TP53 variants did not meet LFS diagnostic/testing criteria while 20.9% (28/134) with confirmed or inferred somatic origins did. TP53 biallelic inactivation occurred in 75% of germline carrier tumors and was also prevalent in other groups, causing an elevated tumor-observed variant allelic fraction (VAF). However, somatic evidence including low VAF correctly identified only 27.8% (25/90) of patients with confirmed somatic TP53 variants. CONCLUSION: The high incidence and variable phenotype of LFS in this cohort highlights the importance of assessing germline status of TP53 variants identified in all pediatric tumors. Without clear somatic evidence, distinguishing somatic from germline origins is challenging. Classifying germline and somatic variants should follow appropriate guidelines.

Machine Learning Methods and Visual Observations to Categorize Behavior of Grazing Cattle Using Accelerometer Signals.

Accelerometers worn by animals produce distinct behavioral signatures, which can be classified accurately using machine learning methods such as random forest decision trees. The objective of this study was to identify accelerometer signal separation among parsimonious behaviors. We achieved this objective by (1) describing functional differences in accelerometer signals among discrete behaviors, (2) identifying the optimal window size for signal pre-processing, and (3) demonstrating the number of observations required to achieve the desired level of model accuracy,. Crossbred steers (Bos taurus indicus; n = 10) were fitted with GPS collars containing a video camera and tri-axial accelerometers (read-rate = 40 Hz). Distinct behaviors from accelerometer signals, particularly for grazing, were apparent because of the head-down posture. Increasing the smoothing window size to 10 s improved classification accuracy (p < 0.05), but reducing the number of observations below 50% resulted in a decrease in accuracy for all behaviors (p < 0.05). In-pasture observation increased accuracy and precision (0.05 and 0.08 percent, respectively) compared with animal-borne collar video observations.

Assessing Endoscopic Response in Locally Advanced Rectal Cancer Treated with Total Neoadjuvant Therapy: Development and Validation of a Highly Accurate Convolutional Neural Network.

BACKGROUND: Rectal tumors display varying degrees of response to total neoadjuvant therapy (TNT). We evaluated the performance of a convolutional neural network (CNN) in interpreting endoscopic images of either a non-complete response to TNT or local regrowth during watch-and-wait surveillance. METHODS: Endoscopic images from stage II/III rectal cancers treated with TNT from 2012 to 2020 at a single institution were retrospectively reviewed. Images were labelled as Tumor or No Tumor based on endoscopy timing (before, during, or after treatment) and the tumor's endoluminal response. A CNN was trained using ResNet-50 architecture. The area under the curve (AUC) was analyzed during training and for two test sets. The main test set included images of tumors treated with TNT. The other contained images of local regrowth. The model's performance was compared to sixteen surgeons and surgical trainees who evaluated 119 images for evidence of tumor. Fleiss' kappa was calculated by respondent experience level. RESULTS: A total of 2717 images from 288 patients were included; 1407 (51.8%) contained tumor. The AUC was 0.99, 0.98, and 0.92 for training, main test, and local regrowth test sets. The model performed on par with surgeons of all experience levels for the main test set. Interobserver agreement was good ( k = 0.71-0.81). All groups outperformed the model in identifying tumor from images of local regrowth. Interobserver agreement was fair to moderate ( k = 0.24-0.52). CONCLUSIONS: A highly accurate CNN matched the performance of colorectal surgeons in identifying a noncomplete response to TNT. However, the model demonstrated suboptimal accuracy when analyzing images of local regrowth.

Comparative Effects of Advanced Footwear Technology in Track Spikes and Road-Racing Shoes on Running Economy.

PURPOSE: Determine the effects of advanced footwear technology (AFT) in track spikes and road-racing shoes on running economy (RE). METHODS: Four racing shoes (3 AFT and 1 control) and 3 track spikes (2 AFT and 1 control) were tested in 9 male distance runners on 2 visits. Shoes were tested in a random sequence over 5-minute trials on visit 1 (7 trials at 16 km·h-1; 5-min rest between trials) and in the reverse/mirrored order on visit 2. Metabolic data were collected and averaged across visits. RESULTS: There were significant differences across footwear conditions for oxygen consumption (F = 13.046; P < .001) and energy expenditure (F = 14.710; P < .001). Oxygen consumption (in milliliters per kilogram per minute) in both the first AFT spike (49.1 [1.7]; P < .001; dz = 2.1) and the other AFT spike (49.3 [1.7]; P < .001; dz = 1.7) was significantly lower than the control spike (50.2 [1.6]), which represented a 2.1% (1.0%) and 1.8% (1.0%) improvement in RE, respectively, for the AFT spikes. When comparing the subjects' most economic shoe by oxygen consumption (49.0 [1.5]) against their most economic spike (49.0 [1.8]), there were no statistical differences (P = .82). Similar statistical conclusions were made when comparing energy expenditure (in watts per kilogram). CONCLUSIONS: AFT track spikes improved RE ∼2% relative to a traditional spike. Despite their heavier mass, AFT shoes resulted in similar RE as AFT spikes. This could make the AFT shoe an attractive option for longer track races, particularly in National Collegiate Athletic Association and high school athletics, where there are no stack-height rules.

ER-associated degradation adapter Sel1L is required for CD8+ T cell function and memory formation following acute viral infection.

The maintenance of antigen-specific CD8+ T cells underlies the efficacy of vaccines and immunotherapies. Pathways contributing to CD8+ T cell loss are not completely understood. Uncovering the pathways underlying the limited persistence of CD8+ T cells would be of significant benefit for developing novel strategies of promoting T cell persistence. Here, we demonstrate that murine CD8+ T cells experience endoplasmic reticulum (ER) stress following activation and that the ER-associated degradation (ERAD) adapter Sel1L is induced in activated CD8+ T cells. Sel1L loss limits CD8+ T cell function and memory formation following acute viral infection. Mechanistically, Sel1L is required for optimal bioenergetics and c-Myc expression. Finally, we demonstrate that human CD8+ T cells experience ER stress upon activation and that ER stress is negatively associated with improved T cell functionality in T cell-redirecting therapies. Together, these results demonstrate that ER stress and ERAD are important regulators of T cell function and persistence.

Oncologic Outcomes of Salvage Abdominoperineal Resection for Anal Squamous Cell Carcinoma Initially Managed with Chemoradiation.

Background: Abdominoperineal resection (APR) has been advocated for persistent or recurrent disease after failure of chemoradiation (CRT) for anal squamous cell cancer (SCC). Treatment with salvage APR can potentially achieve a cure. This study aimed to analyze oncological outcomes for salvage APR in a recent time period at a comprehensive cancer center. Methods: A retrospective review of all patients who underwent APR for biopsy-proven persistent or recurrent anal SCC between 1 January 2007 and 31 December 2020 was performed. Patients with stage IV disease at the time of initial diagnosis and patients with missing data were excluded. Univariate analysis was used with a chi-square test for categorical variables, and non-parametric tests were used for continuous variables. Kaplan-Meier survival analysis was performed to evaluate disease-specific (DSS), post-APR local recurrence-free (RFS), and disease-free survival (DFS). Results: A total of 96 patients were included in the analysis: 39 (41%) with persistent disease and 57 (59%) with recurrent SCC after chemoradiation had been completed. The median follow-up was 22 months (IQR 11-47). Forty-nine patients (51%) underwent extended APR and/or pelvic exenteration. Eight (8%) patients developed local recurrence, 30 (31%) developed local and distant recurrences, and 16 (17%) developed distant recurrences alone. The 3-year DSS, post-APR local recurrence-free survival, and disease-free survival were 53.8% (95% CI 43.5-66.5%), 54.5% (95% CI 44.4-66.8%), and 26.8% (95% CI 18.6-38.7%), respectively. In multivariate logistic regression analysis, positive microscopic margin (OR 10.0, 95% CI 2.16-46.12, p = 0.003), positive nodes in the surgical specimen (OR 9.19, 95% CI 1.99-42.52, p = 0.005), and lymphovascular invasion (OR 2.61 95% CI 1.05-6.51, p = 0.04) were associated with recurrence of disease. Gender, indication for APR (recurrent vs. persistent disease), HIV status, extent of surgery, or type of reconstruction did not influence survival outcomes. Twenty patients had targeted tumor-sequencing data available. Nine patients had PIK3CA mutations, seven of whom experienced a recurrence. Conclusions: Salvage APR for anal SCC after failed CRT was associated with poor disease-specific survival and low recurrence-free survival. Anal SCC patients undergoing salvage APR should be counseled that microscopic positive margins, positive lymph nodes, or the presence of lymphovascular invasion in the APR specimen are prognosticators for disease relapse. Our results accentuate the necessity for additional treatment strategies for the ongoing treatment challenge of persistent or recurrent anal SCC after failed CRT.

Dosimetric validation of intensity-modulated bolus electron conformal therapy planning and delivery using an anthropomorphic cylindrical head phantom.

PURPOSE: This work investigated the dosimetric accuracy of the intensity-modulated bolus electron conformal therapy (IM-BECT) planning and delivery process using the decimal ElectronRT (eRT) treatment planning system. METHODS: An IM-BECT treatment plan was designed using eRT for a cylindrical, anthropomorphic retromolar trigone phantom. Treatment planning involved specification of beam parameters and design of a variable thickness wax bolus and Passive Radiotherapy Intensity Modulator for Electrons (PRIME) device, which was comprised of 33 tungsten island blocks of discrete diameters from 0.158 to 0.223 cm (Intensity Reduction Factors from 0.937 to 0.875, respectively) inside a 10.1 × 6.7 cm2 copper cutout. For comparison of calculation accuracy, a BECT plan was generated by copying the IM-BECT plan and removing the intensity modulation. For both plans, a 16 MeV electron beam was used with 104.7 cm source-to-surface distance to bolus. In-phantom TLD-100 measurements (N = 47) were compared with both eRT planned dose distributions, which used the pencil beam redefinition algorithm with modifications for passive electron intensity modulation (IM-PBRA). Dose difference and distance to agreement (DTA) metrics were computed for each measurement point. RESULTS: Comparison of measured dose distributions with planned dose distributions yielded dose differences (calculated minus measured) characterized by a mean and standard deviation of -0.36% ± 1.64% for the IM-BECT plan, which was similar to -0.36% ± 1.90% for the BECT plan. All dose measurements were within 5% of the planned dose distribution, with both the BECT and IM-BECT measurement sets having 46/47 (97.8%) points within 3% or within 3 mm of the respective treatment plans. CONCLUSIONS: It was found that the IM-BECT treatment plan generated using eRT was sufficiently accurate for clinical use when compared to TLD measurements in a cylindrical, anthropomorphic phantom, and was similarly accurate to the BECT treatment plan in the same phantom.

Pharmacogenomic synthetic lethal screens reveal hidden vulnerabilities and new therapeutic approaches for treatment of NF1-associated tumors.

Neurofibromatosis Type 1 (NF1) is a common cancer predisposition syndrome, caused by heterozygous loss of function mutations in the tumor suppressor gene NF1. Individuals with NF1 develop benign tumors of the peripheral nervous system (neurofibromas), originating from the Schwann cell linage after somatic loss of the wild type NF1 allele, some of which progress further to malignant peripheral nerve sheath tumors (MPNST). There is only one FDA approved targeted therapy for symptomatic plexiform neurofibromas and none approved for MPNST. The genetic basis of NF1 syndrome makes associated tumors ideal for using synthetic drug sensitivity approaches to uncover therapeutic vulnerabilities. We developed a drug discovery pipeline to identify therapeutics for NF1-related tumors using isogeneic pairs of NF1-proficient and deficient immortalized human Schwann cells. We utilized these in a large-scale high throughput screen (HTS) for drugs that preferentially kill NF1-deficient cells, through which we identified 23 compounds capable of killing NF1-deficient Schwann cells with selectivity. Multiple hits from this screen clustered into classes defined by method of action. Four clinically interesting drugs from these classes were tested in vivo using both a genetically engineered mouse model of high-grade peripheral nerve sheath tumors and human MPNST xenografts. All drugs tested showed single agent efficacy in these models as well as significant synergy when used in combination with the MEK inhibitor selumetinib. This HTS platform yielded novel therapeutically relevant compounds for the treatment of NF1-associated tumors and can serve as a tool to rapidly evaluate new compounds and combinations in the future.

Iron regulates the quiescence of naive CD4 T cells by controlling mitochondria and cellular metabolism.

In response to an immune challenge, naive T cells undergo a transition from a quiescent to an activated state acquiring the effector function. Concurrently, these T cells reprogram cellular metabolism, which is regulated by iron. We and others have shown that iron homeostasis controls proliferation and mitochondrial function, but the underlying mechanisms are poorly understood. Given that iron derived from heme makes up a large portion of the cellular iron pool, we investigated iron homeostasis in T cells using mice with a T cell-specific deletion of the heme exporter, FLVCR1 [referred to as knockout (KO)]. Our finding revealed that maintaining heme and iron homeostasis is essential to keep naive T cells in a quiescent state. KO naive CD4 T cells exhibited an iron-overloaded phenotype, with increased spontaneous proliferation and hyperactive mitochondria. This was evidenced by reduced IL-7R and IL-15R levels but increased CD5 and Nur77 expression. Upon activation, however, KO CD4 T cells have defects in proliferation, IL-2 production, and mitochondrial functions. Iron-overloaded CD4 T cells failed to induce mitochondrial iron and exhibited more fragmented mitochondria after activation, making them susceptible to ferroptosis. Iron overload also led to inefficient glycolysis and glutaminolysis but heightened activity in the hexosamine biosynthetic pathway. Overall, these findings highlight the essential role of iron in controlling mitochondrial function and cellular metabolism in naive CD4 T cells, critical for maintaining their quiescent state.

Disordered clock protein interactions and charge blocks turn an hourglass into a persistent circadian oscillator.

Organismal physiology is widely regulated by the molecular circadian clock, a feedback loop composed of protein complexes whose members are enriched in intrinsically disordered regions. These regions can mediate protein-protein interactions via SLiMs, but the contribution of these disordered regions to clock protein interactions had not been elucidated. To determine the functionality of these disordered regions, we applied a synthetic peptide microarray approach to the disordered clock protein FRQ in Neurospora crassa. We identified residues required for FRQ's interaction with its partner protein FRH, the mutation of which demonstrated FRH is necessary for persistent clock oscillations but not repression of transcriptional activity. Additionally, the microarray demonstrated an enrichment of FRH binding to FRQ peptides with a net positive charge. We found that positively charged residues occurred in significant "blocks" within the amino acid sequence of FRQ and that ablation of one of these blocks affected both core clock timing and physiological clock output. Finally, we found positive charge clusters were a commonly shared molecular feature in repressive circadian clock proteins. Overall, our study suggests a mechanistic purpose for positive charge blocks and yielded insights into repressive arm protein roles in clock function.

Association of Lateral Pelvic Lymph Nodes with Disease Recurrence and Organ Preservation in Patients with Distal Rectal Adenocarcinoma Treated with Total Neoadjuvant Therapy.

OBJECTIVE: Assess the significance of enlarged lateral lymph nodes (LLN) for disease recurrence, metastasis, and organ preservation in patients with rectal cancer. BACKGROUND: Optimal treatment of rectal adenocarcinoma involving LLN is subject to debate. METHODS: A post hoc analysis of the OPRA trial, a multicenter study of patients with rectal cancer treated with total neoadjuvant therapy (TNT) followed by total mesorectal excision or watch-and-wait management. We analyzed the association of visible LLN (LLN+), LLN≥7 mm (short axis) on baseline MRI, and LLN≥4 mm on restaging MRI with recurrence, metastasis, and rectum preservation. RESULTS: At baseline, 57 out of 324 (18%) patients had LLN+. In 30 (53%) of 57 patients with LLN+ on baseline MRI, the LLN disappeared after TNT. Disease recurrence in LLN was rare (3.5% of patients with LLN+ and 0.4% of patients with LLN-). All patients with recurrence in LLN also had distant metastasis. The rate of organ preservation was significantly lower in patients with LLN≥4 mm on restaging MRI (P=0.013). We found no significant differences in rates of local recurrence or metastasis between patients with LLN+ vs. LLN- and in patients with LLN≥7 vs.<7 mm on baseline MRI. LLN dissection was performed in 3 patients; 2 of them died of distant metastasis. CONCLUSIONS: LLN involvement is not associated with disease recurrence or metastasis, but persistence of LLN≥4 mm after TNT is negatively associated with rectum preservation in patients with locally advanced rectal cancer treated with TNT. Dissection of lateral nodes likely benefits few patients.

Update on Cancer Predisposition Syndromes and Surveillance Guidelines for Childhood Brain Tumors.

Tumors of the central nervous system (CNS) comprise the second most common group of neoplasms in childhood. The incidence of germline predisposition among children with brain tumors continues to grow as our knowledge on disease etiology increases. Some children with brain tumors may present with nonmalignant phenotypic features of specific syndromes (e.g., nevoid basal cell carcinoma syndrome, neurofibromatosis type 1 and type 2, DICER1 syndrome, and constitutional mismatch-repair deficiency), while others may present with a strong family history of cancer (e.g., Li-Fraumeni syndrome) or with a rare tumor commonly found in the context of germline predisposition (e.g., rhabdoid tumor predisposition syndrome). Approximately 50% of patients with a brain tumor may be the first in a family identified to have a predisposition. The past decade has witnessed a rapid expansion in our molecular understanding of CNS tumors. A significant proportion of CNS tumors are now well characterized and known to harbor specific genetic changes that can be found in the germline. Additional novel predisposition syndromes are also being described. Identification of these germline syndromes in individual patients has not only enabled cascade testing of family members and early tumor surveillance but also increasingly affected cancer management in those patients. Therefore, the AACR Cancer Predisposition Working Group chose to highlight these advances in CNS tumor predisposition and summarize and/or generate surveillance recommendations for established and more recently emerging pediatric brain tumor predisposition syndromes.