FUS Regulates Activity of MicroRNA-Mediated Gene Silencing.

MicroRNA-mediated gene silencing is a fundamental mechanism in the regulation of gene expression. It remains unclear how the efficiency of RNA silencing could be influenced by RNA-binding proteins associated with the microRNA-induced silencing complex (miRISC). Here we report that fused in sarcoma (FUS), an RNA-binding protein linked to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), interacts with the core miRISC component AGO2 and is required for optimal microRNA-mediated gene silencing. FUS promotes gene silencing by binding to microRNA and mRNA targets, as illustrated by its action on miR-200c and its target ZEB1. A truncated mutant form of FUS that leads its carriers to an aggressive form of ALS, R495X, impairs microRNA-mediated gene silencing. The C. elegans homolog fust-1 also shares a conserved role in regulating the microRNA pathway. Collectively, our results suggest a role for FUS in regulating the activity of microRNA-mediated silencing.

An intra-tumoral niche maintains and differentiates stem-like CD8 T cells.

Tumour-infiltrating lymphocytes are associated with a survival benefit in several tumour types and with the response to immunotherapy1-8. However, the reason some tumours have high CD8 T cell infiltration while others do not remains unclear. Here we investigate the requirements for maintaining a CD8 T cell response against human cancer. We find that CD8 T cells within tumours consist of distinct populations of terminally differentiated and stem-like cells. On proliferation, stem-like CD8 T cells give rise to more terminally differentiated, effector-molecule-expressing daughter cells. For many T cells to infiltrate the tumour, it is critical that this effector differentiation process occur. In addition, we show that these stem-like T cells reside in dense antigen-presenting-cell niches within the tumour, and that tumours that fail to form these structures are not extensively infiltrated by T cells. Patients with progressive disease lack these immune niches, suggesting that niche breakdown may be a key mechanism of immune escape.

Prostate Health Index density improves detection of clinically significant prostate cancer.

OBJECTIVES: To explore the utility of Prostate Health Index (PHI) density for the detection of clinically significant prostate cancer (PCa) in a contemporary cohort of men presenting for diagnostic evaluation of PCa. PATIENTS AND METHODS: The study cohort included patients with elevated prostate-specific antigen (PSA; >2 ng/mL) and negative digital rectal examination who underwent PHI testing and prostate biopsy at our institution in 2015. Serum markers were prospectively measured per standard clinical pathway. PHI was calculated as ([{-2}proPSA/free PSA] × [PSA]½ ), and density calculations were performed using prostate volume as determined by transrectal ultrasonography. Logistic regression was used to assess the ability of serum markers to predict clinically significant PCa, defined as any Gleason score ≥7 cancer or Gleason score 6 cancer in >2 cores or >50% of any positive core. RESULTS: Of 118 men with PHI testing who underwent biopsy, 47 (39.8%) were found to have clinically significant PCa on biopsy. The median (interquartile range [IQR]) PHI density was 0.70 (0.43-1.21), and was 0.53 (0.36-0.75) in men with negative biopsy or clinically insignificant PCa and 1.21 (0.74-1.88) in men with clinically significant PCa (P < 0.001). Clinically significant PCa was detected in 3.6% of men in the first quartile of PHI density (<0.43), 36.7% of men in the IQR of PHI density (0.43-1.21), and 80.0% of men with PHI density >1.21 (P < 0.001). Using a threshold of 0.43, PHI density was 97.9% sensitive and 38.0% specific for clinically significant PCa, and 100% sensitive for Gleason score ≥7 disease. Compared with PSA (area under the curve [AUC] 0.52), PSA density (AUC 0.70), %free PSA (AUC 0.75), the product of %free PSA and prostate volume (AUC 0.79), and PHI (AUC 0.76), PHI density had the highest discriminative ability for clinically significant PCa (AUC 0.84). CONCLUSIONS: Based on the present prospective single-centre experience, PHI density could be used to avoid 38% of unnecessary biopsies, while failing to detect only 2% of clinically significant cancers.

Infectious complications in transfused patients after radical cystectomy.

INTRODUCTION: Infectious complications are common after radical cystectomy (RC), and allogeneic blood transfusions may increase infection risk by an immunosuppressive effect. While it has been suggested that perioperative blood transfusion (PBT) may be associated with adverse oncologic outcomes after RC, no large analyses have assessed whether PBT increases the risk of perioperative infection after RC. MATERIALS AND METHODS: We used the Nationwide Inpatient Sample (1998 to 2011) to study the rate of PBT during RC for bladder cancer and identify infectious complications. We compared rates of infectious complications in patients who did and did not receive PBT and developed a multivariable model to assess the independent risk of infectious complication associated with PBT controlling for age, year of surgery, obesity, chronic kidney disease, comorbidity score, and type of urinary diversion. RESULTS: We identified 126,454 RCs performed during the study period. A total of 34,203 (27%) received a PBT. The use of PBT increased over the study period, from 18.4% in 1998 to 31.6% in 2011 (p < 0.0001). Patients who received a PBT had an increased risk of perioperative infectious complications [36.7% versus 27.7%, unadjusted OR (95% CI) = 1.51 (1.43-1.60), p < 0.0001]. After adjusting for potential confounders, PBT remained an independent predictor of infectious complications [adjusted OR (95% CI) = 1.46 (1.38-1.55), p < 0.0001]. CONCLUSIONS: This analysis provides strong observational evidence that PBT is associated with an increased risk of perioperative infectious complications, which may be secondary to transfusion-related immunomodulation. Urologists should aggressively pursue blood conservation strategies and adhere to evidence-based restrictive transfusion thresholds, particularly given the rising rate of PBT.

Topline/Final Diagnostic Inclusion of Relevant Histologic Findings in Surgical Pathology Reporting of Carcinoma in Prostate Biopsies.

INTRODUCTION: As the list of histologic parameters to include in surgical pathology reports of prostate cancer biopsies grows, some pathologists include this information in the microscopic description or summary sections of the report, whereas others include it in the "topline" or final diagnosis section. This prompted us to develop a multi-institutional survey to assess reporting trends among genitourinary (GU) pathologists. METHODS: A survey instrument was shared among 110 GU pathologists via surveymonkey.com. Anonymized respondent data was analyzed. RESULTS: Eighty-four (76%) participants completed the survey across four continents. Most participants report tumor volume quantitation (88%), number of cores involved (89%), and both Gleason grade and Grade group (93%) in their topline; 71% include percent of pattern 4, with another 16% including it depending on cancer grade; 58% include the presence of cribriform growth pattern 4, with another 11% including it depending on cancer grade. When present, most include extraprostatic extension (90%), prostatic intraductal carcinoma (77%), and perineural invasion (77%). Inclusion of atypical intraductal proliferation (AIP) in the topline diagnosis was cancer grade-dependent, with 74% including AIP in Grade group 1, 61% in Grade group 2, 45% in Grade group 3, 30% in Grade group 4, and 26% in Grade group 5 cancers. CONCLUSION: Certain histologic features such as Gleason grade and tumor volume/cores involved are frequently included in the topline diagnosis, whereas the incorporation of other findings are more variably included. Prostate biopsy reporting remains a dynamic process with stylistic similarities and differences existing among GU pathologists.

World Health Organization 2022 Classification Update: Radiologic and Pathologic Features of Papillary Renal Cell Carcinomas.

RATIONALE AND OBJECTIVES: To describe imaging and pathology features of newly defined papillary renal cell carcinoma (pRCC) based on the WHO 2022 update. MATERIALS AND METHODS: This retrospective study included 87 patients with 93 pathologically proven papillary renal cell carcinomas who underwent pre-treatment renal mass protocol CT or MRI. Baseline and post-treatment follow-up imaging was evaluated by two radiologists systematically based on established lexicon. RESULTS: At pathology, 63 (68%) were grade 1-2, 29 (31%) were grade 3-4, and 1 (%) was unreported. At surgical pathology, 84 (90%) were localized (≤pT2b), 5 (5%) were pT3a, and none were ≥pT3b; 4 (4%) had unknown pT stage (core biopsies). 33 (35%) had necrosis and 39 (41%) had hemorrhage. None had sarcomatoid or rhabdoid differentiation. At imaging, 73 (83%) were solid and 16 (17%) were cystic. Of 16 cystic masses, four were Bosniak class IIF (three were heterogeneously T1 hyperintense) and 12 were class IV. All were well-circumscribed. 92 (99%) were hypovascular. Median follow-up for 74 patients was 30 months (IQR 12-56). One untreated patient had non-regional nodal metastasis at presentation, and one patient had metastasis to lymph nodes and bones after surgery, but the patient had unresected renal masses elsewhere without pathology. Otherwise, no recurrence or metastases were detected. CONCLUSION: Most pRCCs present as a hypovascular, circumscribed, solid renal mass. A few pRCCs present as the newly defined Bosniak class IIF subtype. Our results can form the basis of a non-invasive, likelihood score to identify this relatively indolent pathology in the era of virtual biopsy and active surveillance.

TDP-43 neurotoxicity and protein aggregation modulated by heat shock factor and insulin/IGF-1 signaling.

TAR DNA-binding protein 43 (TDP-43) plays a key role in the neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The nature of the TDP-43-mediated neurotoxicity associated with these diseases is not yet understood. Here, we have established transgenic Caenorhabditis elegans models that express human TDP-43 variants in the nervous system, including the full-length wild-type (WT) and mutant proteins and a pathologic C-terminal fragment. The C. elegans models developed severe locomotor defects associated with the aggregation of TDP-43 in neurons. In comparison to parallel Cu/Zn superoxide dismutase worm models, transgenic full-length TDP-43, including the WT protein, was highly neurotoxic. In addition, TDP-43 demonstrated an unusually high tendency to aggregate, a property intrinsic to the WT protein. The C-terminal 25 kDa fragment of TDP-43 was unstable but remarkably aggregation-prone. Distinct disulfide-linked TDP-43 dimers and oligomers were detected. In C. elegans, the neurotoxicity and the protein aggregation of TDP-43 were regulated by environmental temperature and heat shock transcriptional factor 1, indicating that a deficiency in protein quality control is a risk factor for TDP-43 proteinopathy. Furthermore, the neurotoxicity and the protein aggregation of TDP-43 can be significantly attenuated by a deficiency in the insulin/insulin-like growth factor 1 (IGF-1) signaling in C. elegans and mammalian cells. These results suggest that protein misfolding underlies the aging-dependent neurodegeneration associated with TDP-43 and that the insulin/IGF-1 signaling may be a target for therapies.

Clinicopathologic analysis of patients undergoing repeat transurethral resection of bladder tumour following an initial diagnosis of urothelial carcinoma with lamina propria invasion and variant/divergent histology.

AIMS: A subset of patients with urothelial carcinoma (UCa) and lamina propria (LP) invasion in bladder biopsies/transurethral resections (TURs) are at significant risk for recurrence and have increased rates of progression to UCa with muscularis propria (MP) invasion. The clinicopathologic features of this patient population has not been well characterised in the Pathology literature. METHODS: We performed a search through our urologic pathology files and expert consult cases of the senior author for bladder biopsies/TURs of UCa with LP invasion and variant/divergent histology from 2014 to 2020. Patients with a prior diagnosis of UCa with MP invasion or upper tract UCa were excluded. Clinicopathologic data were obtained. RESULTS: Ninety-five patients with at least one biopsy/TUR of UCa with LP invasion and variant/divergent histology were identified. Mean patient age was 72 years (range: 46-92 years) with a male predominance 2.3:1. Initial variant/divergent histologies identified were: glandular (35.8%), squamous (23.2%), micropapillary (20%), clear cell/lipid rich (12.6%), diffuse/signet ring/plasmacytoid (10.5%), nested (9.5%), sarcomatoid (6.3%), poorly differentiated/anaplastic (4.2%), small cell (2.1%), lymphoepithelioma-like (2.1%), osteoclast-like giant cells (1.1%) and tumour giant cells (1.1%). Two or more variant histologies were identified in 18.9% of these cases. The rate of micropapillary UCa was significantly higher in multifocal tumours compared with unifocal tumours (37% vs 7.1%). CONCLUSIONS: In our cohort of patients undergoing early repeat biopsy/TUR, 75% of patients had persistent UCa. Additionally, almost 25% of patients had a prior diagnosis of UCa without a variant/divergent histology identified. Our findings highlight the critical role of repeat biopsy/TUR especially in a subset of patients who have variant/divergent histology, even in the absence of MP invasion.

Does Milan affect management? A retrospective analysis of resection rate and time to surgery among Milan categories.

INTRODUCTION: The Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) is an established system with reproducible risk of malignancies (ROM) for salivary gland fine needle aspiration (SGFNA). No studies have reviewed the relationship between Milan categories and the resection rate (RR) and time to resection (TTR). METHODS: We searched our database (January 1, 2011 to January 4, 2021) for non-lymphoma SGFNAs and assigned appropriate MSRSGC categories. RR and TTR were calculated and compared for each category. A literature search was performed; RRs and TTRs were compared. RESULTS: Seven hundred and eighty SGFNAs were identified, 333 with follow-up. RR was highest in suspicious for malignancy (SUS, V; 70.6%, n = 12/17), followed by the salivary gland neoplasm of uncertain malignant potential (SUMP, IVb; 69.6%, n = 80/115) and malignant (M, VI; 55.6%, n = 75/135). Among M, primary tumors had a higher RR (65.1%, n = 41/63) than metastases (47.2%, n = 34/72, p = .36). In literature review, SUS had the highest RR (69.3%, n = 233/336) followed by M (61.6%, n = 821/1332) and SUMP (60.2%, n = 632/1050). TTR was shorter in SUS (mean = 32.3 days, median = 25 days). Within the benign neoplasms (BN, IVa), Pleomorphic adenomas (PAs) had a higher RR than Warthin tumors (WTs) (66.3% vs. 37.2%, p < .00001), and a shorter TTR (median = 63 days vs. 90 days). CONCLUSIONS: Tumors classified as SUS had higher RR and at shorter intervals than those classified as SUMP. PAs have higher RRs and more expedient surgery than WTs. Cases classified as M are less likely to undergo follow-up than SUS, perhaps due to a lower RR for metastases.

Risk of malignancy in T1-hyperintense Bosniak version 2019 class II and IIF cystic renal masses.

BACKGROUND: Bosniak classification version 2019 includes cystic masses in class II and IIF based partly on their hyperintense appearance at T1-weighted MRI. The prevalence of malignancy in non-enhancing heterogeneously T1-hyperintense masses is unknown, nor whether the pattern of T1 hyperintensity affects malignancy likelihood. PURPOSE: To determine the malignancy proportion among six patterns of T1 hyperintensity within non-enhancing cystic renal masses. METHODS: This retrospective, single-institution study included 72 Bosniak class II and IIF, non-enhancing, T1-hyperintense cystic renal masses. Diagnosis was confirmed by histopathology or by follow-up imaging demonstrating 5-year size and morphologic stability, decreased in size by ≥ 30%, resolution, or Bosniak down-classification. Six patterns of T1 hyperintensity were pre-defined: homogeneous (pattern A), fluid-fluid level (pattern B), peripherally markedly T1-hyperintense (pattern C), containing a T1-hyperintense non-enhancing nodule (pattern D), peripherally T1-hypointense (pattern E), and heterogeneously T1-hyperintense without a distinct pattern (pattern F). Three readers independently assigned each mass to a pattern. Individual and mean malignancy proportion were determined. Mann-Whitney test and Fischer's exact test compared the likelihood of malignancy between patterns. Inter-reader agreement was analyzed with Gwet's agreement coefficient (AC). RESULTS: Among 72 masses, the mean number of masses assigned was 11 (15%) to pattern A, 21 (29%) to pattern B, 6 (8%) to pattern C, 7 (10%) to pattern D, 5 (7%) to pattern E, and 22 (31%) to pattern F. Five of 72 masses (7%) were malignant; none was assigned pattern A, B, or D. Mean malignancy proportion was 5% (0/9, 1/6, and 0/4) for pattern C, 13% (0/4, 1/3, and 1/7) for pattern E, and 18% (5/20, 3/21, and 4/25) for pattern F. Malignant masses were more likely assigned to pattern E or F (p = 0.003-0.039). Inter-reader agreement was substantial (Gwet's AC: 0.68). CONCLUSION: Bosniak version 2019 class IIF masses that are non-enhancing and heterogeneously T1-hyperintense with a fluid-fluid level are likely benign. Those that are non-enhancing and heterogeneously T1-hyperintense without a distinct pattern have a malignancy proportion up to 25% (5/20).

Nondiagnostic salivary gland FNAs are associated with decreased risk of malignancy compared with "all-comer" patients: Analysis of the Milan System for Reporting Salivary Gland Cytopathology with a focus on Milan I: Nondiagnostic.

BACKGROUND: The Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) reports a 25% rate of malignancy (ROM) for the Milan I: Nondiagnostic (ND) category. We clarify the ROM of ND salivary gland fine-needle aspirations (SGFNAs) based on our institutional experience and review of the literature. METHODS: Overall risk of malignancy (OROM) and that for those with surgical/flow cytometric follow-up (FROM) for each category and "all-comers" were calculated for Emory SGFNAs from January 2010 through March 2021. From a literature review of 50 articles using MSRSGC, distribution of diagnoses, rates of follow-up, FROM, and OROM by category were calculated. FROMs and OROMs between ND FNAs and all-comers were compared. Milan I rate was compared with the ratio of Milan I OROM to all-comer OROM. RESULTS: Of 819 SGFNAs at Emory, 12.8% (n = 105/819) were ND. Thirty-two had known follow-up, with 12 (37.5%) being malignant. Nonmucinous cyst contents accounted for 26.7% of ND SGFNAs (n = 28/105); all 7 with surgical follow-up were benign. Of 50 MSRSGC studies, 18.2% (n = 2384/13,129) of SGFNAs were classified as ND, 26.6% (n = 635/2384) with known follow-up. Total FROM and OROM for ND FNAs (15.7% and 4.1%, respectively) were significantly lower than those for all-comers (24.9% and 11.4%, respectively) (p < .001). There was no relationship between rate of ND SGFNA and ND ROM. CONCLUSIONS: The ND category is associated with a lower ROM than that of all-comer SGFNA patients. The "true" ROM for ND SGFNAs is likely best estimated by the 4.1% OROM. SGFNAs showing nonmucinous cyst contents have a particularly low ROM. Rate of ND SGFNAs does not influence ND ROM.

Clinical outcome following checkpoint therapy in renal cell carcinoma is associated with a burst of activated CD8 T cells in blood.

PURPOSE: Checkpoint therapy is now the cornerstone of treatment for patients with renal cell carcinoma (RCC) with advanced disease, but biomarkers are lacking to predict which patients will benefit. This study proposes potential immunological biomarkers that could developed for predicting therapeutic response in patients with RCC. METHODS: Using flow cytometry, RNA sequencing, and T-cell receptor (TCR) sequencing, we investigated changes in T cells in the peripheral blood of patients with advanced RCC after receiving immunotherapy. We used immunofluorescence (IF) imaging and flow cytometry to investigate how intratumoral T cells in patients' tumors (resected months/years prior to receiving checkpoint therapy) predicted patient outcomes after immunotherapy. RESULTS: We found that a small proportion of CD4 and CD8 T cells in the blood activate following checkpoint therapy, expressing the proliferation marker Ki67 and activation markers HLA-DR and CD38. Patients who had the highest increase in these HLA-DR +CD38+CD8 T cells after treatment had the best antitumor immune response and experienced clinical benefit. Using RNA sequencing, we found that while these cells expanded in most patients, their phenotype did not drastically change during treatment. However, when we analyzed the TCR repertoire of these HLA-DR +CD38+CD8+T cells, we found that only patients who clinically benefitted had a burst of new clonotypes enter this pool of activated cells. Finally, we found that abundant T cells in the untreated tumors predicted clinical benefit to checkpoint therapy on disease progression. CONCLUSIONS: Together, these data suggest that having a strong pre-existing immune response and immediate peripheral T-cell activation after checkpoint therapy is a predictor of clinical benefit in patients with RCC.

Combining immune check-point blockade and cryoablation in an immunocompetent hormone sensitive murine model of prostate cancer.

BACKGROUND: Prostate cancer remains the second leading cause of cancer related death in men. Immune check point blocking antibodies have revolutionized treatment of multiple solid tumors, but results in prostate cancer remain marginal. Previous reports have suggested that local therapies, in particular cryoablation might increase tumor immunogenicity. In this work, we examine potential synergism between tumor cryoabalation and check point blocking antibodies. METHODS: FVB/NJ mice were injected subcutaneously into each flank with either 1 × 106 or 0.2 × 106 isogenic hormone sensitive Myc-Cap cells to establish synchronous grafts. Mice were treated with four intraperitoneal injections of anti-PD-1 (10 mg/kg), anti-CTLA-4 (1 mg/kg), or isotype control antibody with or without adjuvant cryoablation of the larger tumor graft and with or without neo-adjuvant androgen deprivation with degarelix (ADT). Mouse survival and growth rates of tumor grafts were measured. The immune dependency of observed oncological effects was evaluated by T cell depletion experiments. RESULTS: Treatment with anti-CTLA-4 antibody and cryoablation delayed the growth of the distant tumor by 14.8 days (p = 0.0006) and decreased the mortality rate by factor of 4 (p = 0.0003) when compared to cryoablation alone. This synergy was found to be dependent on CD3+ and CD8+ cells. Combining PD-1 blockade with cryoablation did not show a benefit over use of either treatment alone. Addition of ADT to anti-PD1 therapy and cryoablation doubled the time to accelerated growth in the untreated tumors (p = 0.0021) and extended survival when compared to cryoablation combined with ADT in 25% of the mice. Effects of combining anti-PD1 with ADT and cryoablation on mouse survival were obviated by T cell depletion. CONCLUSION: Trimodal therapy consisting of androgen deprivation, cryoablation and PD-1 blockade, as well as the combination of cryoablation and low dose anti-CTLA-4 blockade showed that local therapies with cryoablation could be considered to augment the effects of checkpoint blockade in prostate cancer.