Analysis of phosphofructokinase-1 activity as affected by pH and ATP concentration.

A key player in energy metabolism is phosphofructokinase-1 (PFK1) whose activity and behavior strongly influence glycolysis and thus have implications in many areas. In this research, PFK1 assays were performed to convert F6P and ATP into F-1,6-P and ADP for varied pH and ATP concentrations. PFK1 activity was assessed by evaluating F-1,6-P generation velocity in two ways: (1) directly calculating the time slope from the first two or more datapoints of measured product concentration (the initial-velocity method), and (2) by fitting all the datapoints with a differential equation explicitly representing the effects of ATP and pH (the modeling method). Similar general trends of inhibition were shown by both methods, but the former gives only a qualitative picture while the modeling method yields the degree of inhibition because the model can separate the two simultaneous roles of ATP as both a substrate of reaction and an inhibitor of PFK1. Analysis based on the model suggests that the ATP affinity is much greater to the PFK1 catalytic site than to the inhibitory site, but the inhibited ATP-PFK1-ATP complex is much slower than the uninhibited PFK1-ATP complex in product generation, leading to reduced overall reaction velocity when ATP concentration increases. The initial-velocity method is simple and useful for general observation of enzyme activity while the modeling method has advantages in quantifying the inhibition effects and providing insights into the process.

Fully Automated Deep Learning Model to Detect Clinically Significant Prostate Cancer at MRI.

Background Multiparametric MRI can help identify clinically significant prostate cancer (csPCa) (Gleason score ≥7) but is limited by reader experience and interobserver variability. In contrast, deep learning (DL) produces deterministic outputs. Purpose To develop a DL model to predict the presence of csPCa by using patient-level labels without information about tumor location and to compare its performance with that of radiologists. Materials and Methods Data from patients without known csPCa who underwent MRI from January 2017 to December 2019 at one of multiple sites of a single academic institution were retrospectively reviewed. A convolutional neural network was trained to predict csPCa from T2-weighted images, diffusion-weighted images, apparent diffusion coefficient maps, and T1-weighted contrast-enhanced images. The reference standard was pathologic diagnosis. Radiologist performance was evaluated as follows: Radiology reports were used for the internal test set, and four radiologists' PI-RADS ratings were used for the external (ProstateX) test set. The performance was compared using areas under the receiver operating characteristic curves (AUCs) and the DeLong test. Gradient-weighted class activation maps (Grad-CAMs) were used to show tumor localization. Results Among 5735 examinations in 5215 patients (mean age, 66 years ± 8 [SD]; all male), 1514 examinations (1454 patients) showed csPCa. In the internal test set (400 examinations), the AUC was 0.89 and 0.89 for the DL classifier and radiologists, respectively (P = .88). In the external test set (204 examinations), the AUC was 0.86 and 0.84 for the DL classifier and radiologists, respectively (P = .68). DL classifier plus radiologists had an AUC of 0.89 (P < .001). Grad-CAMs demonstrated activation over the csPCa lesion in 35 of 38 and 56 of 58 true-positive examinations in internal and external test sets, respectively. Conclusion The performance of a DL model was not different from that of radiologists in the detection of csPCa at MRI, and Grad-CAMs localized the tumor. © RSNA, 2024 Supplemental material is available for this article. See also the editorial by Johnson and Chandarana in this issue.

Racial, ethnic, and socioeconomic disparities in rates of stage IV prostate cancer after USPSTF category "D" recommendation against prostate-specific antigen screening: a retrospective cohort study.

Background: In 2012 the United States Preventative Services Task Force (USPSTF) changed its prostate-specific antigen (PSA) screening recommendation to a category "D". The purpose of this study is to examine racial, ethnic, and socioeconomic differences in risk of presentation with metastatic prostate cancer (mPCa) at time of diagnosis before and after the 2012 USPSTF category "D" recommendation. Methods: This is a population-based cohort study. We identified patients with mPCa at diagnosis within the National Cancer Database from 2004-2017. Logistic regression models were used to examine associations of mPCa with age, race, ethnicity, geographic location, education level, income, and insurance status. Linear regression models assuming underlying binomial distribution were fitted to annual percentage of mPCa at diagnosis for years 2012-2017 to evaluate the post category "D" recommendation era. Results: From 2004 to 2017, 88,987 patients presented with mPCa. A higher percentage of mPCa was noted post-USPSTF category "D" recommendation, with a disproportionately greater increase observed among Hispanics and non-Hispanic Blacks [Δslope/year: Hispanics (0.0092), non-Hispanic Blacks (0.0073) and non-Hispanic Whites (0.0070)]. Insurance status impacts race/ethnicity differently: uninsured Hispanics were 3.66 times more likely to present with mPCa than insured Hispanics, while uninsured non-Hispanic Blacks were 2.62 times more likely to present with mPCa than insured non-Hispanic Blacks. Household income appears to be associated with differences in mPCa, particularly among non-Hispanic Blacks. Those earning <$30,000 were more likely to present with mPCa compared to higher income brackets. Conclusions: Since the USPSTF grade "D" recommendation against PSA screening, the percentage of mPCa at diagnosis has increased, with a higher rate of increase among Hispanic and non-Hispanic Blacks compared to non-Hispanic Whites.

The ACROBAT 2022 challenge: Automatic registration of breast cancer tissue.

The alignment of tissue between histopathological whole-slide-images (WSI) is crucial for research and clinical applications. Advances in computing, deep learning, and availability of large WSI datasets have revolutionised WSI analysis. Therefore, the current state-of-the-art in WSI registration is unclear. To address this, we conducted the ACROBAT challenge, based on the largest WSI registration dataset to date, including 4,212 WSIs from 1,152 breast cancer patients. The challenge objective was to align WSIs of tissue that was stained with routine diagnostic immunohistochemistry to its H&E-stained counterpart. We compare the performance of eight WSI registration algorithms, including an investigation of the impact of different WSI properties and clinical covariates. We find that conceptually distinct WSI registration methods can lead to highly accurate registration performances and identify covariates that impact performances across methods. These results provide a comparison of the performance of current WSI registration methods and guide researchers in selecting and developing methods.

Spatial analysis of recurrent glioblastoma reveals perivascular niche organization.

Tumor evolution is driven by genetic variation; however, it is the tumor microenvironment (TME) that provides the selective pressure contributing to evolution in cancer. Despite high histopathological heterogeneity within glioblastoma (GBM), the most aggressive brain tumor, the interactions between the genetically distinct GBM cells and the surrounding TME are not fully understood. To address this, we analyzed matched primary and recurrent GBM archival tumor tissues with imaging-based techniques aimed to simultaneously evaluate tumor tissues for the presence of hypoxic, angiogenic, and inflammatory niches, extracellular matrix (ECM) organization, TERT promoter mutational status, and several oncogenic amplifications on the same slide and location. We found that the relationships between genetic and TME diversity are different in primary and matched recurrent tumors. Interestingly, the texture of the ECM, identified by label-free reflectance imaging, was predictive of single-cell genetic traits present in the tissue. Moreover, reflectance of ECM revealed structured organization of the perivascular niche in recurrent GBM, enriched in immunosuppressive macrophages. Single-cell spatial transcriptomics further confirmed the presence of the niche-specific macrophage populations and identified interactions between endothelial cells, perivascular fibroblasts, and immunosuppressive macrophages. Our results underscore the importance of GBM tissue organization in tumor evolution and highlight genetic and spatial dependencies.

Inhibition of pyruvate dehydrogenase accelerates anaerobic glycolysis under postmortem simulating conditions.

This research aimed to explore the potential influence of mitochondria on the rate of anaerobic glycolysis. We hypothesized that mitochondria could reduce the rate of anaerobic glycolysis and pH decline by metabolizing a portion of glycolytic pyruvate. We utilized an in vitro model and incorporated CPI-613 and Avidin to inhibit pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC), respectively. Four treatments were tested: 400 µM CPI-613, 1.5 U/ml Avidin, 400 µM CPI-613 + 1.5 U/ml Avidin, or control. Glycolytic metabolites and pH of the in vitro model were evaluated throughout a 1440-min incubation period. CPI-613-containing treatments, with or without Avidin, decreased pH levels and increased glycogen degradation and lactate accumulation compared to the control and Avidin treatments (P < 0.05), indicating increased glycolytic flux. In a different experiment, two treatments, 400 µM CPI-613 or control, were employed to track the fates of pyruvate using [13C6]glucose. CPI-613 reduced the contribution of glucose carbon to tricarboxylic acid cycle intermediates compared to control (P < 0.05). To test whether the acceleration of acidification in reactions containing CPI-613 was due to an increase in the activity of key enzymes of glycogenolysis and glycolysis, we evaluated the activities of glycogen phosphorylase, phosphofructokinase, and pyruvate kinase in the presence or absence of 400 µM CPI-613. The CPI-613 treatment did not elicit an alteration in the activity of these three enzymes. These findings indicate that inhibiting PDH increases the rate of anaerobic glycolysis and pH decline, suggesting that mitochondria are potential regulators of postmortem metabolism.

Virtual alignment of pathology image series for multi-gigapixel whole slide images.

Interest in spatial omics is on the rise, but generation of highly multiplexed images remains challenging, due to cost, expertise, methodical constraints, and access to technology. An alternative approach is to register collections of whole slide images (WSI), generating spatially aligned datasets. WSI registration is a two-part problem, the first being the alignment itself and the second the application of transformations to huge multi-gigapixel images. To address both challenges, we developed Virtual Alignment of pathoLogy Image Series (VALIS), software which enables generation of highly multiplexed images by aligning any number of brightfield and/or immunofluorescent WSI, the results of which can be saved in the ome.tiff format. Benchmarking using publicly available datasets indicates VALIS provides state-of-the-art accuracy in WSI registration and 3D reconstruction. Leveraging existing open-source software tools, VALIS is written in Python, providing a free, fast, scalable, robust, and easy-to-use pipeline for registering multi-gigapixel WSI, facilitating downstream spatial analyses.

Tranexamic Acid Was Not Associated With a Reduction in Bleeding Complications Related to Holmium Laser Enucleation of the Prostate.

OBJECTIVE: To compare outcomes in males given perioperative tranexamic acid (TXA) during holmium laser enucleation of the prostate (HoLEP) to a historical cohort that did not receive TXA. METHODS: Our cohort included HoLEP patients (N = 1037) from August 2018 through November 2022. Exclusion criteria included history of stroke, transient ischemic attack, or coronary stent placed within 18 months. The primary predictor was TXA during HoLEP. The primary outcome variable was a return to the operating room (RTOR) for bleeding. Secondary outcomes included catheter reinsertion, transfusion, and clotting complications. Multivariable models evaluating associations of TXA with outcomes were adjusted for potential confounders. RESULTS: In our cohort, 214/1035 had one or more complications with 81 having a Clavien-Dindo Grade of 3a or higher, 114/1037 with catheter reinsertion, 36/1037 RTOR for bleeding, and 15/1035 had a transfusion. TXA use was not associated with 30-day RTOR. 3.7% of those without TXA had a RTOR, and 3.1% of those with TXA had a RTOR (adjusted odds ratio 0.63, 95% CI 0.28-1.38, P = .25). Transfusion rates were higher for those without TXA (2.0%) compared to with (0.5%). This was statistically significant after adjustment (adjusted odds ratio 0.13, 95% CI 0.03-0.69, P = .016) and after correction for multiple testing (corrected P = .048). TXA use was not associated with catheter reinsertion or clotting complications. CONCLUSION: Routine perioperative intravenous TXA in HoLEP patients was not associated with a reduction of RTOR for bleeding, catheter reinsertion, or clotting complications. TXA was associated with decreased transfusions with a low overall event rate of 1.4%.

Early apical release versus En-bloc no touch technique for holmium laser enucleation of the prostate: a high-volume single-surgeon cohort study.

PURPOSE: To determine whether the early apical release (EAR) technique for holmium laser enucleation of the prostate (HOLEP) is associated with improved perioperative outcomes compared to the En-bloc no touch (EBNT) technique. METHODS: Consecutive men treated with HOLEP by a single surgeon from August 2018 to March 2021 were identified. Beginning in June 2021 all procedures were performed using the EAR technique, and these were compared to the preceding cases done with the EBNT technique. Intraoperative outcomes included operative time, need for open conversion, enucleation efficiency (tissue removed per minute of OR time), and enucleation ratio (tissue removed relative to preoperative gland size on imaging). Postoperative outcomes included catheter reinsertion, blood transfusion, and complications classified by the Clavien-Dindo scale. RESULTS: We identified 801 men, including 571 (71%) treated with EBNT and 230 (29%) with EAR. Median preoperative characteristics were similar between groups. The EAR approach was associated with significantly longer mean operating room time, 100.5 min versus 91.9 min, p = 0.003. However, EAR patients had a much lower rate of conversion to open cystotomy (0.43% versus 3.0%). There were no significant differences in rate of catheter reinsertion or perioperative complications between groups (p > 0.05). CONCLUSION: EAR technique by an experienced HoLEP surgeon resulted in longer operative times, potentially reflecting an initial learning curve, but essentially eliminated the need for open cystotomy. Perioperative results including catheter reinsertion rate and bleeding complications were similar between the two cohorts. These data support continued use of the EAR technique for HOLEP to minimize risk of open conversion.

Urinary Outcomes After Magnetic Resonance Imaging-Guided Whole-Gland Transurethral Ultrasound Ablation for Prostate Cancer: Comparison of Suprapubic Tube to Indwelling Urethral Catheter.

Background: MRI-guided transurethral ultrasound ablation (TULSA) is under investigation for whole-gland ablation of low- and intermediate-risk prostate cancer. The ideal method for post-TULSA bladder drainage through postoperative suprapubic tube (SPT) vs indwelling urethral catheter (UC) has not been established. The objective of this study was to evaluate urinary outcomes after whole-gland TULSA, comparing postoperative SPT with UC. Materials and Methods: Two-institution retrospective analysis of whole-gland TULSA for men with grade group 1 and 2 prostate cancer. One institution placed SPT at the time of TULSA with clamp trials (day 10) and removal once voiding. The second placed UC until void trial (day 7). Outcomes included the International Prostate Symptom Score (IPSS), urinary bother score, catheter reinsertion, stricture, clean intermittent catheterization (CIC), and incontinence. Results: Forty-five patients (median age 67) were analyzed. The UC cohort (N = 26) was older (p = 0.007) than the SPT cohort (N = 19) but with similar baseline prostate volumes, IPSS, and urinary bother scores. Patients receiving UC had fewer days with catheter (p = 0.013). Although UC patients suffered more lower urinary tract symptoms at 1-month post-TULSA, there was no significant difference between IPSS scores at baseline and 6 months after surgery regardless of urinary management strategy, although the UC group noted significantly decreased urinary bother. Rates of infection were similar between groups. Six strictures were observed overall, with more in the SPT group, although the difference was not significant (4/19 [21.1%] SPT; 2/26 [7.7%] UC). At 6 months, incontinence rates were low and similar between groups (2/19 [10.5%] SPT; 4/26 [15.4%] UC) and only one patient (UC) required CIC. Conclusions: Our overall findings suggest that SPT and UC are both acceptable options for postoperative bladder drainage after whole-gland TULSA, with statistically similar rates of urinary complications but a slightly different side effect profile.

Prostatic urethral lift (PUL) clip causing postoperative pain after holmium laser enucleation of the prostate (HoLEP).

Few series exist in the literature of holmium laser enucleation of the prostate (HoLEP) after prostatic urethral lift (PUL). Even less well known are potential complications seen after a patient undergoes PUL followed by HoLEP. We present our case of a unique clinical finding of a PUL clip and suture found in the urethra of a patient after HoLEP.

Spectro 1-A Potential Spectrophotometer for Measuring Color and Myoglobin Forms in Beef.

The objective of this study was to compare the Color Muse Spectro 1 sensor to the HunterLab MiniScan XE Plus spectrophotometer for evaluating beef color. Color coordinates (lightness (L*), redness (a*), yellowness (b*), chroma (C*), and hue (h*)), myoglobin redox forms (metmyoglobin (MMb), deoxymyoglobin (DMb), and oxymyoglobin (OMb)), and metmyoglobin reducing ability (MRA) were measured on beef steaks over a 5-days storage period. The results indicated that L*, b*, C*, MMb%, OMb%, and MRA% values obtained with Spectro 1 were comparable to those of MiniScan. However, Spectro 1 values for a* were overestimated compared to MiniScan (p < 0.05), whereas those for h* and DMb% were underestimated (p < 0.05). Regardless, Spectro 1 had the capability to detect changes in color coordinates, myoglobin forms, and MRA throughout the storage period. Bland−Altman plots demonstrated that L*, b*, and C* are interchangeable between the two instruments, but it was not the case for a*, h*, myoglobin forms, and MRA. Color coordinates measured by Spectro 1 exhibited excellent stability over time, evidenced by the low total color difference (ΔE*ab) values. Collectively, these findings indicate that Spectro 1 is a potential alternative spectrophotometer for studying meat color and myoglobin redox forms.

Cattle breed type and anabolic implants impact calpastatin expression and abundance of mRNA associated with protein turnover in the longissimus thoracis of feedlot steers.

Two methods that the beef cattle industry can use to improve efficiency, sustainability, and economic viability are growth promotants and crossbreeding cattle of different breed types. In the United States, over 90% of cattle receive an anabolic implant at some point during production resulting in an overall increase in skeletal muscle growth. Recent research suggests that the two main cattle breed types, Bos indicus and Bos taurus, respond differently to anabolic implants. The objective of this study was to characterize changes that occur in skeletal muscle following implanting in Bos indicus influenced steers or Bos taurus steers. Twenty steers were stratified by initial weight in a 2 × 2 factorial design examining two different breeds: Angus (AN; n = 10) or Santa Gertrudis influenced (SG; n = 10), and two implant strategies: no implant (CON; n = 10) or a combined implant containing 120 mg TBA and 24 mg E2 (IMP; n = 10; Revalor-S, Merck Animal Health). Skeletal muscle biopsies were taken from the longissimus thoracis (LT) 2 and 10 d post-implantation. The mRNA abundance of 24 genes associated with skeletal muscle growth were examined, as well as the protein expression of µ-calpain and calpastatin. Succinate dehydrogenase mRNA abundance was impacted (P = 0.05) by a breed × treatment interaction 2 d post-implanting, with SG-CON having a greater increased abundance than all other steers. A tendency for a breed × treatment interaction was observed for calpain-6 mRNA (P = 0.07), with SG-CON having greater abundance than AN-CON and SG-IMP. Additionally, calpastatin protein expression was altered (P = 0.01) by a breed × treatment interaction, with SG-CON and SG-IMP steers having increased expression (P = 0.01) compared with AN-CON steers. At 2 d post-implanting, a breed × treatment interaction was observed with SG-CON steers having greater (P = 0.05) mRNA abundance of mitogen-activated protein kinase compared with AN-CON steers. Furthermore, breed affected (P = 0.05) calpastatin abundance with AN steers having increased (P = 0.05) abundance 2 d post-implanting compared with SG steers. Meanwhile, implants tended to affect (P = 0.09) muscle RING finger protein-1 mRNA abundance, with CON steers having increased (P = 0.09) abundance compared with that of IMP steers. These findings suggest that cattle breed type and anabolic implants impact calpastatin expression and mRNA abundance associated with protein turnover in the LT of feedlot steers 2 and 10 d post-implantation.

Two methods that the beef cattle industry can use to potentially improve efficiency, sustainability, and economic viability are growth promotants and crossbreeding cattle of different breed types. In the United States, over 90% of cattle receive at least one anabolic implant during the production cycle resulting in improvements in production and overall economic and environmental sustainability. Research suggests that the two main cattle breed types, Bos indicus and Bos taurus, respond differently to different anabolic implant strategies. The objective of this study was to characterize changes that occur in the skeletal muscle following implanting in Bos indicus influenced animals and Bos taurus animals. This research measured mRNA abundance of 24 genes associated with skeletal muscle growth, and protein expression of calpain-1 and calpastatin. The findings of this research suggest that anabolic implants and cattle breed type interact to cause changes in mRNA abundance in the longissimus thoracis that are related to protein turnover of skeletal muscle. Furthermore, calpastatin protein abundance was also altered by this breed × treatment interaction. This research demonstrates that anabolic implants cause molecular changes in skeletal muscle of feedlot steers, with some of these changes being breed dependent.

Impact of Severe Acute Respiratory Syndrome Coronavirus 2-Induced COVID-19 on Fixed Operating Room Times in Urologic Operations.

Objective: To evaluate the impact of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, on operating room (OR) efficiency for urologic procedures using the concept of fixed OR times. Patients and Methods: Over a 24-month period, urology OR data were prospectively collected. Operations were divided into fixed and variable time points. The fixed OR times were in-roomw to anesthesia-release time, anesthesia-release to cut time, in-room to cut time, and close to wheels-out time. Data from January 1, 2019, to December 31, 2019, were pre-COVID-19 data, and data from April 1, 2020, to December 31, 2020, were post-COVID-19 data. Operations were grouped into endoscopic, implant, major open, and robotic-assisted cases. In the post-COVID-19 era, all patients had a negative polymerase chain reaction test result within 48 hours of operation. The Wilcoxon rank sum test was used to compare the fixed OR times between the pre- and post-COVID-19 eras. Results: A total of 3189 procedures were evaluated: 2058 endoscopic operations (1124 in the pre-COVID-19 era and 934 in the post-COVID-19 era), 343 implant procedures (192 in the pre-COVID-19 era and 151 in the post-COVID-19 era), 222 major open procedures (119 in the pre-COVID-19 era and 103 in the post-COVID-19 era), and 566 robotic-assisted procedures (338 in the pre-COVID-19 era and 228 in the post-COVID-19 era). There were no fixed OR times in any of the examined groups that were negatively impacted by COVID-19. The percentage of the total OR time occupied by fixed OR variables in the pre-COVID-19 era was 40.6% for endoscopic operations, 41.1% for implant procedures, 29.8% for major open procedures, and 21.8% for robotic-assisted procedures. Conclusion: A substantial portion of the total OR time includes fixed time points. Furthermore, COVID-19 did not have a negative impact on fixed OR times in a negative testing environment. Urologic OR efficiency should be maintained in the post-COVID-19 era.

Analysis of Holmium Laser Enucleation of Prostate Fixed Operating Room Times.

OBJECTIVE: To evaluate factors influencing fixed operating room time during holmium laser enucleation of the prostate. MATERIALS AND METHODS: A prospective observational study was performed for all holmium laser enucleation of the prostate (HoLEP) cases performed by a single surgeon over a 24-month period. Operating room (OR) time was divided into fixed and variable time. The variable time was defined as cut-to-close time. Fixed time included in room time to anesthesia release time (IRAT), anesthesia release time to cut time (ARCT), and close time to wheels out (CTWO). The effects of time of day and anesthesia personnel (AP) changes on fixed operating room time were evaluated. RESULTS: A total of 406 HoLEPs were analyzed. There was no statistically significant difference in nonprocedural OR times between morning and afternoon surgeries (IRAT, P = .38, ARCT P = .10, CTWO P = .77). Median nonprocedural OR times accounted for 27% (IQR: 22%-31%) of the total procedure time in the AM group and 29% (IQR: 24%-33%) in the PM group (P = .005). Of the HoLEPs,78.1% (178/228) experienced one or more AP changes during the procedure. The median fixed OR time was not significantly different between procedures with 1 AP and procedures with ≥2 APs (IRAT, P = .53; ARCT, P = .71; CTWO, P = .98). CONCLUSION: Fixed operating room time makes up a significant portion of HoLEP procedures and should be considered when evaluating OR efficiency. The time of day and number of anesthesia personnel involved did not affect the fixed OR times.

Immunosuppressive niche engineering at the onset of human colorectal cancer.

The evolutionary dynamics of tumor initiation remain undetermined, and the interplay between neoplastic cells and the immune system is hypothesized to be critical in transformation. Colorectal cancer (CRC) presents a unique opportunity to study the transition to malignancy as pre-cancers (adenomas) and early-stage cancers are frequently resected. Here, we examine tumor-immune eco-evolutionary dynamics from pre-cancer to carcinoma using a computational model, ecological analysis of digital pathology data, and neoantigen prediction in 62 patient samples. Modeling predicted recruitment of immunosuppressive cells would be the most common driver of transformation. As predicted, ecological analysis reveals that progressed adenomas co-localized with immunosuppressive cells and cytokines, while benign adenomas co-localized with a mixed immune response. Carcinomas converge to a common immune "cold" ecology, relaxing selection against immunogenicity and high neoantigen burdens, with little evidence for PD-L1 overexpression driving tumor initiation. These findings suggest re-engineering the immunosuppressive niche may prove an effective immunotherapy in CRC.

Ovarian cancer immunogenicity is governed by a narrow subset of progenitor tissue-resident memory T cells.

Despite repeated associations between T cell infiltration and outcome, human ovarian cancer remains poorly responsive to immunotherapy. We report that the hallmarks of tumor recognition in ovarian cancer-infiltrating T cells are primarily restricted to tissue-resident memory (TRM) cells. Single-cell RNA/TCR/ATAC sequencing of 83,454 CD3+CD8+CD103+CD69+ TRM cells and immunohistochemistry of 122 high-grade serous ovarian cancers shows that only progenitor (TCF1low) tissue-resident T cells (TRMstem cells), but not recirculating TCF1+ T cells, predict ovarian cancer outcome. TRMstem cells arise from transitional recirculating T cells, which depends on antigen affinity/persistence, resulting in oligoclonal, trogocytic, effector lymphocytes that eventually become exhausted. Therefore, ovarian cancer is indeed an immunogenic disease, but that depends on ∼13% of CD8+ tumor-infiltrating T cells (∼3% of CD8+ clonotypes), which are primed against high-affinity antigens and maintain waves of effector TRM-like cells. Our results define the signature of relevant tumor-reactive T cells in human ovarian cancer, which could be applicable to other tumors with unideal mutational burden.

Alternative RNA splicing defects in pediatric cancers: new insights in tumorigenesis and potential therapeutic vulnerabilities.

BACKGROUND: Compared with adult cancers, pediatric cancers are uniquely characterized by a genomically stable landscape and lower tumor mutational burden. Alternative splicing, however, a global cellular process that produces different messenger RNA/protein isoforms from a single messenger RNA transcript, has been increasingly implicated in the development of pediatric cancers. DESIGN: We review the current literature on the role of alternative splicing in adult cancer, cancer predisposition syndromes, and pediatric cancers. We also describe multiple splice variants identified in adult cancers and confirmed through comprehensive genomic profiling in our institutional cohort of rare, refractory, and relapsed pediatric and adolescent young adult cancer patients. Finally, we summarize the contributions of alternative splicing events to neoantigens and chemoresistance and prospects for splicing-based therapies. RESULTS: Published dysregulated splicing events can be categorized as exon inclusion, exon exclusion, splicing factor up-regulation, or splice site alterations. We observe these phenomena in cancer predisposition syndromes (Lynch syndrome, Li-Fraumeni syndrome, CHEK2) and pediatric leukemia (B-cell acute lymphoblastic leukemia), sarcomas (Ewing sarcoma, rhabdomyosarcoma, osteosarcoma), retinoblastoma, Wilms' tumor, and neuroblastoma. Within our institutional cohort, we demonstrate splice variants in key regulatory genes (CHEK2, TP53, PIK3R1, MDM2, KDM6A, NF1) that resulted in exon exclusion or splice site alterations, which were predicted to impact functional protein expression and promote tumorigenesis. Differentially spliced isoforms and splicing proteins also impact neoantigen creation and treatment resistance, such as imatinib or glucocorticoid regimens. Additionally, splice-altering strategies with the potential to change the therapeutic landscape of pediatric cancers include antisense oligonucleotides, adeno-associated virus gene transfers, and small molecule inhibitors. CONCLUSIONS: Alternative splicing plays a critical role in the formation and growth of pediatric cancers, and our institutional cohort confirms and highlights the broad spectrum of affected genes in a variety of cancers. Further studies that elucidate the mechanisms of disease-inducing splicing events will contribute toward the development of novel therapeutics.

IgA-Dominated Humoral Immune Responses Govern Patients' Outcome in Endometrial Cancer.

Recent studies suggest that B cells could play an important role in the tumor microenvironment. However, the role of humoral responses in endometrial cancer remains insufficiently investigated. Using a cohort of 107 patients with different histological subtypes of endometrial carcinoma, we evaluated the role of coordinated humoral and cellular adaptive immune responses in endometrial cancer. Concomitant accumulation of T, B, and plasma cells at tumor beds predicted better survival. However, only B-cell markers corresponded with prolonged survival specifically in high-grade endometrioid type and serous tumors. Immune protection was associated with class-switched IgA and, to a lesser extent, IgG. Expressions of polymeric immunoglobulin receptor (pIgR) by tumor cells and its occupancy by IgA were superior predictors of outcome and correlated with defects in methyl-directed DNA mismatch repair. Mechanistically, pIgR-dependent, antigen-independent IgA occupancy drove activation of inflammatory pathways associated with IFN and TNF signaling in tumor cells, along with apoptotic and endoplasmic reticulum stress pathways, while thwarting DNA repair mechanisms. Together, these findings suggest that coordinated humoral and cellular immune responses, characterized by IgA:pIgR interactions in tumor cells, determine the progression of human endometrial cancer as well as the potential for effective immunotherapies. SIGNIFICANCE: This study provides new insights into the crucial role of humoral immunity in human endometrial cancer, providing a rationale for designing novel immunotherapies against this prevalent malignancy. See related commentary by Osorio and Zamarin, p. 766.

Utilizing Preoperative Magnetic Resonance Imaging to Self-assess Enucleation Ratio in Holmium Laser Enucleation of the Prostate.

OBJECTIVE: To determine if transition zone (TZ) volume as measured by magnetic resonance imaging (MRI) correlates closely with specimen weight and postoperative prostate-specific antigen (PSA) and to determine if this correlation improved with increased experience. We hypothesize that information from a preoperative MRI can be leveraged to self-assess competency in holmium laser enucleation of the prostate (HoLEP). METHODS: From August 2018 to June 2021 567 men were identified who underwent HoLEP by a single surgeon and had their data entered into a database. Ninety-seven men were identified who had an MRI in our image archives. A radiologist blinded to the specimen weight measured the transition zone (TZ) using image analysis software. TZ volume was then correlated with specimen weight for the entire study period and subdivided into four consecutive periods. Preoperative and Postoperative PSA data was analyzed. RESULTS: Seventeen percent of men who underwent HoLEP at our institution had an MRI preoperatively. The TZ to total prostate volume ratio was 70%. The overall correlation coefficient between TZ volume and specimen weight was 0.914 (P-value <.001). There was no statistically significant improvement in correlation coefficient over time when divided into four equal subgroups. Median PSA reduction was 91% (7.9-0.7 ng/dL). CONCLUSION: Preoperative MRI derived TZ volume correlates closely with specimen weight. We present a reference cohort from a high volume HoLEP center to facilitate HoLEP learners to self-assess competency. Lack of increased correlation over time is likely due to an experienced HoLEP surgeon who is on the flatter portion of his learning curve.