True Mitotic Count Prediction in Gastrointestinal Stromal Tumors: Bayesian Network Model and PROMETheus (Preoperative Mitosis Estimator Tool) Application Development.

BACKGROUND: Gastrointestinal stromal tumors (GISTs) present a complex clinical landscape, where precise preoperative risk assessment plays a pivotal role in guiding therapeutic decisions. Conventional methods for evaluating mitotic count, such as biopsy-based assessments, encounter challenges stemming from tumor heterogeneity and sampling biases, thereby underscoring the urgent need for innovative approaches to enhance prognostic accuracy. OBJECTIVE: The primary objective of this study was to develop a robust and reliable computational tool, PROMETheus (Preoperative Mitosis Estimator Tool), aimed at refining patient stratification through the precise estimation of mitotic count in GISTs. METHODS: Using advanced Bayesian network methodologies, we constructed a directed acyclic graph (DAG) integrating pertinent clinicopathological variables essential for accurate mitotic count prediction on the surgical specimen. Key parameters identified and incorporated into the model encompassed tumor size, location, mitotic count from biopsy specimens, surface area evaluated during biopsy, and tumor response to therapy, when applicable. Rigorous testing procedures, including prior predictive simulations, validation utilizing synthetic data sets were employed. Finally, the model was trained on a comprehensive cohort of real-world GIST cases (n=80), drawn from the repository of the Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Humanitas Research Hospital, with a total of 160 cases analyzed. RESULTS: Our computational model exhibited excellent diagnostic performance on synthetic data. Different model architecture were selected based on lower deviance and robust out-of-sample predictive capabilities. Posterior predictive checks (retrodiction) further corroborated the model's accuracy. Subsequently, PROMETheus was developed. This is an intuitive tool that dynamically computes predicted mitotic count and risk assessment on surgical specimens based on tumor-specific attributes, including size, location, surface area, and biopsy-derived mitotic count, using posterior probabilities derived from the model. CONCLUSIONS: The deployment of PROMETheus represents a potential advancement in preoperative risk stratification for GISTs, offering clinicians a precise and reliable means to anticipate mitotic counts on surgical specimens and a solid base to stratify patients for clinical studies. By facilitating tailored therapeutic strategies, this innovative tool is poised to revolutionize clinical decision-making paradigms, ultimately translating into improved patient outcomes and enhanced prognostic precision in the management of GISTs.

Disruptions in antigen processing and presentation machinery on sarcoma.

BACKGROUND: The antigen processing machinery (APM) plays a critical role in generating tumor-specific antigens that can be recognized and targeted by the immune system. Proper functioning of APM components is essential for presenting these antigens on the surface of tumor cells, enabling immune detection and destruction. In many cancers, defects in APM can lead to immune evasion, contributing to tumor progression and poor clinical outcomes. However, the status of the APM in sarcomas is not well characterized, limiting the development of effective immunotherapeutic strategies for these patients. METHODS: We investigated 126 patients with 8 types of bone and soft tissue sarcoma operated between 2001-2021. Tissue microarrays mapped 11 specific areas in each case. The presence/absence of APM protein was determined through immunohistochemistry. Bayesian networks were used. RESULTS: All investigated sarcomas had some defects in APM. The least damaged component was HLA Class I subunit ß2-microglobulin and HLA Class II. The proteasome LMP10 subunit was defective in leiomyosarcoma (LMS), myxoid liposarcoma (MLPS), and dedifferentiated liposarcoma (DDLPS), while MHC I transporting unit TAP2 was altered in undifferentiated pleomorphic sarcoma (UPS), gastrointestinal stromal tumor (GIST), and chordoma (CH). Among different neoplastic areas, high-grade areas showed different patterns of expression compared to high lymphocytic infiltrate areas. Heterogeneity at the patient level was also observed. Loss of any APM component was prognostic of distant metastasis (DM) for LMS and DDLPS and of overall survival (OS) for LMS. CONCLUSION: Sarcomas exhibit a high degree of defects in APM components, with differences among histotypes and tumoral areas. The most commonly altered APM components were HLA Class I subunit ß2-microglobulin, HLA Class I subunit α (HC10), and MHC I transporting unit TAP2. The loss of APM components was prognostic of DM and OS and clinically relevant for LMS and DDLPS. This study explores sarcoma molecular mechanisms, enriching personalized therapeutic approaches.

A Propensity Score-matched Comparison of Micro-ultrasound-guided Transrectal and Magnetic Resonance Imaging/Transrectal Ultrasound Fusion-guided Transperineal Prostate Biopsies for Detection of Clinically Significant Prostate Cancer.

Background and objective: High-resolution micro-ultrasound (microUS) is an advanced imaging tool. Our objective was to determine whether systematic microUS use for transrectal biopsy (TRBx) improves the detection rate for clinically significant prostate cancer (csPCa) in comparison to transperineal biopsy (TPBx) performed with magnetic resonance imaging (MRI)/conventional transrectal ultrasound (TRUS) fusion software. Methods: We retrospectively analyzed data for men who underwent prostate biopsies, including those on active surveillance (AS). TRBx was performed under microUS guidance, while MRI/TRUS fusion was consistently used to guide TPBx. Patients were matched according to propensity score matching (PSM). The primary endpoint was comparison of the csPCa detection rate with the two approaches. Secondary endpoints included predictors of csPCa (International Society of Urological Pathology grade group ≥2, assessed via multivariable logistic regression) and complication rates. Key findings and limitations: Overall, 1423 patients were enrolled. After applying PSM we identified an analytical cohort of 1094 men, 582 in the TRBx group and 512 in the TPBx group. There was no significant difference in the csPCa detection rate between the TRBx (45%) and TPBx (51%) groups (p = 0.07). Complications occurred in nine of 1094 patients (1%). On adjusted multivariable analysis, TPBx had a similar csPCa detection rate to TRBx (adjusted odds ratio [aOR] 1.26;p = 0.09). Predictors of csPCa detection were a positive family history (aOR 1.68; 95% confidence interval [CI] 1.20-2.35; p = 0.002); age (aOR 1.04, 95% CI 1.02-1.06; p < 0.001); positive digital rectal examination (aOR 2.35, 95% CI 1.70-3.25; p < 0.001); prostate-specific antigen density ≥0.15 ng/ml/cm3 (aOR 3.23, 95% CI 2.47-4.23; p < 0.001); and a Prostate Imaging-Reporting and Data System score ≥3 (aOR 2.46; 95% CI 1.83-3.32; p < 0.001). Limitations include the retrospective nature of the study, the risk of underestimating the complication rate, and the heterogeneity of biopsy indications. Conclusions and clinical implications: TRBx using microUS alone showed a comparable csPCa detection rate to TPBx guided by MRI/TRUS fusion software. Given the better visualization and real-time detection of suspicious zones with microUS, the potential for improvement in the csPCa detection rate with greater integration of microUS in the TPBx setting warrants further investigation. Patient summary: We compared the ability of two different prostate biopsy approaches to detect clinically significant prostate cancer. We found that transrectal biopsy guided by micro-ultrasound had similar detection rates to transperineal biopsy guided by a combination of magnetic resonance imaging and conventional ultrasound. More research is needed to confirm the potential of micro-ultrasound for transperineal biopsy.

Prognostic value of mitotic count in leiomyosarcoma: A comprehensive monocentric retrospective study.

BACKGROUND: Leiomyosarcomas (LMSs) include heterogeneous entities with different clinical courses not entirely predicted by known prognostic factors. In particular, the value of mitotic count as independent prognostic factor in LMS has been poorly investigated. METHODS: We retrospectively analyzed all patients with a diagnosis of LMS who accessed to our Institution from June 1999 to May 2022 for which mitotic count was numerically expressed within the pathology report. Univariate and multivariate analyses were conducted to explore the prognostic value of mitotic count along with other clinical and histological variables. RESULTS: We identified 121 eligible patients, with a median follow-up of 91.03 months (range 0.62-275.2 months). Median progression-free survival (mPFS) was 16.7 months, and median overall survival (mOS) was 105.6 months. In univariate analysis, mitotic count showed a significant impact on PFS and OS, with an hazard ratio per mitotic unit of 1.03 (1.01-1.04, p < 0.001) and 1.03 (1.01-1.04, p = 0.007), respectively. Similar results were found for locally advanced and metastatic patients, separately. Other significant prognostic factors for PFS were stage at diagnosis, performance status, tumor size and Ki-67, while differentiation, necrosis, grade, stage at diagnosis, tumor size, performance status and age at diagnosis were identified for OS. In multivariate analysis, the only significant factors were mitotic count and the presence of metastases at diagnosis for PFS, whereas the same two factors plus age at diagnosis were identified for OS. CONCLUSION: Mitotic count represented the most important histological prognostic factor for OS and PFS in localized and metastatic LMS.

Multidisciplinary management of adolescents and young adults (AYA) sarcoma: A successful effort of an adult high-volume cancer center.

INTRODUCTION: The aim of this retrospective study was to investigate the clinicopathological characteristics of AYA sarcomas and their clinical outcomes at a high-volume single center. METHODS: Demographic, clinicopathological data on the diagnosis, treatment and follow-up of all sarcoma patients aged 16-39 years (ys) observed at our Institute between January 2010 and December 2021 were retrospectively collected, including diagnostic (TTD) and treatment delay(TTT), clinical outcomes (OS and PFS), and late-treatment effects. RESULTS: We identified 228 AYA patients, median age 30 years, 29% ≤ 25 years, 57% males, 88% soft tissue sarcomas (STS), and 12% bone sarcomas (BS). Among STSs, 13% were small round cell tumors (SRCT), 52% intermediate-high-grade, 24% low-grade STSs. Among BS, 32% were high-grade. Median TTD and TTT were 120 (0-8255) and 7 days (0-83), respectively. Surgery was performed in 83%, radiotherapy in 29%, and systemic therapy in 27%. Median follow-up was 72.9 months(1.6-145), 5-year and 10-year OS were 78.5% and 62%, respectively. Kaplan-Meyer analysis showed a significantly better 5-year OS and PFS for patients with >92 days of TTD (OS 85.7% vs. 66.7%, p = 0.001, PFS 50.2% vs. 24.9%, p = 0.009). According to age (≤25 years vs. > 25 years), 5-year OS was 69.8% versus 82.2%, respectively (p = 0.047). CONCLUSION: Our analysis confirmed previous data on sarcoma AYA patients followed in a referral center. Unexpectedly, diagnostic delay was not associated with poor OS and PFS. Patients <25 years showed a poorer prognosis due to the higher incidence of SRCT.

Evaluation of Semen Self-Sampling Yield Predictors and CTC Isolation by Multi-Color Flow Cytometry for Liquid Biopsy of Localized Prostate Cancer.

Liquid biopsy (LB) for prostate cancer (PCa) detection could represent an alternative to biopsy. Seminal fluid (SF) is a source of PCa-specific biomarkers, as 40% of ejaculate derives from the prostate. We tested the feasibility of an SF-based LB by evaluating the yield of semen self-sampling in a cohort of >750 patients with clinically localized PCa. The overall SF collection yield was 18.2% (39% when considering only compliant patients), with about a half of the patients (53.15%) not consenting to SF donation. Independent favorable predictors for SF collection were younger age and lower prostate volume. We implemented a protocol to enrich prostate-derived cells by multi-color flow cytometry and applied it on SF and urine samples from 100 patients. The number of prostate-enriched cells (SYTO-16+ PSMA+ CD45-) was variable, with higher numbers of cells isolated from SF than urine (p value < 0.001). Putative cancer cells (EpCAMhigh) were 2% of isolated cells in both specimens. The fraction of EpCAMhigh cells over prostate-enriched cells (PSMA+) significantly correlated with patient age in both semen and urine, but not with other clinical parameters, such as Gleason Score, ISUP, or TNM stage. Hence, enumeration of prostate-derived cells is not sufficient to guide PCa diagnosis; additional molecular analyses to detect patient-specific cancer lesions will be needed.

Clinical and pathological predictors of persistent T1 HG at second resection.

BACKGROUND: T1 high-grade (HG) non-muscle invasive bladder cancer (NMIBC) has a significant risk of recurrence and progression, and the European Association of Urology recommends a second transurethral resection of the bladder (ReTUR). Stage at ReTUR has been shown to be a reliable predictor of survival, therefore, we sought to assess clinical and pathological predictors associated with the persistence of T1 at ReTUR in our retrospective multicentric cohort. METHODS: This is a retrospective multicentric study of T1 HG patients at transurethral resection of the bladder (TURB) who underwent subsequent ReTUR. All histological samples were sub-classified according to Rete Oncologica Lombarda (ROL) T1 sub-staging system. RESULTS: One hundred and sixty-six patients were enrolled. Forty-four (26.5%) had T1 HG tumor at ReTUR while 93 (56%) had residual tumor of any stage. Lesion size was significantly greater in T1 HG patients at ReTUR, as well as the prevalence of multifocality. The multivariable logistic regression model showed lesion dimension and multifocality as predictors of T1 HG at ReTUR, after adjusting for significant covariables (CIS and detrusor muscle presence). ROL sub-staging system was not a significant predictor, but ROL2 prevalence was higher in the T1 HG at ReTUR group. CONCLUSIONS: Lesion size and multifocality were independent predictors of T1 HG persistence at ReTUR, and patients at risk should be promptly identified and treated accordingly. Our results could help physicians make patient-tailored decisions by identifying those most likely to benefit from a second resection.

Diagnostic performance of microUltrasound at MRI-guided confirmatory biopsy in patients under active surveillance for low-risk prostate cancer.

BACKGROUND: Active surveillance (AS) represents a standard of care of low-risk prostate cancer (PCa). However, the identification and monitoring of AS candidates remains challenging. Microultrasound (microUS) is a novel high-resolution imaging modality for transrectal ultrasonography (TRUS). We explored the impact of microUS TRUS and targeted biopsies in mpMRI-guided confirmatory biopsies. METHODS: Between October 2017 and September 2021, we prospectively enrolled 100 patients scheduled for MRI-guided confirmatory biopsy at 1 year from diagnosis of ISUP 1 PCa. TRUS was performed using the ExactVu microUS system; PRI-MUS protocol was applied to identify suspicious lesions (i.e., PRIMUS score ≥ 3). All patients received targeted biopsies of any identified microUS and mpMRI lesions and complementary systematic biopsies. The proportion of patients upgraded to clinically significant PCa (defined as ISUP ≥ 2 cancer; csPCa) at confirmatory biopsies was determined, and the diagnostic performance of microUS and mpMRI were compared. RESULTS: Ninety-two patients had a suspicious MRI lesion classified PI-RADS 3, 4, and 5 in respectively 28, 16, and 18 patients. MicroUS identified 82 patients with suspicious lesions, classified as PRI-MUS 3, 4, and 5 in respectively 20, 50, and 12 patients, while 18 individuals had no lesions. Thirty-four patients were upgraded to ISUP ≥ 2 cancer and excluded from AS. MicroUS and mpMRI showed a sensitivity of 94.1% and 100%, and an NPV of 88.9% and 100%, respectively, in detecting ISUP ≥ 2 patients. A microUS-mandated protocol would have avoided confirmatory biopsies in 18 patients with no PRI-MUS ≥ 3 lesions at the cost of missing four upgraded patients. CONCLUSIONS: MicroUS and mpMRI represent valuable imaging modalities showing high sensitivity and NPV in detecting csPCa, thus allowing their use for event-triggered confirmatory biopsies in AS patients. MicroUS offers an alternative imaging modality to mpMRI for the identification and real-time targeting of suspicious lesions in AS patients.

Diagnostic Performance and Clinical Impact of PSMA PET/CT versus mpMRI in Patients with a High Suspicion of Prostate Cancer and Previously Negative Biopsy: A Prospective Trial (PROSPET-BX).

BACKGROUND: This prospective single-arm study is designed to compare in parallel 68Ga-PSMA PET/TRUS (transrectal or transperineal) fusion biopsy ("experimental test") with multiparametric MRI (mpMRI)/TRUS fusion prostate biopsy ("standard test") in men with a high suspicion of prostate cancer (PCa) after at least one negative biopsy. The primary objective was to evaluate the diagnostic performance of 68Ga-PSMA PET/TRUS fusion prostate biopsy in comparison to mpMRI/TRUS fusion prostate biopsy analyzed in parallel. Secondarily, we aimed to determine the relationship between the "experimental test" and the histopathological characteristics of the specimen, along with the clinical utility of the "experimental test" compared to the "standard test." SUMMARY: To test the superiority of 68Ga-PSMA PET/CT compared to mpMRI, we will enroll a minimum cohort of 128 patients. Inclusion criteria comprise: age >18 years; blood PSA level >4.0 ng/mL; free-to-total PSA ratio <20%; progressive rise of PSA levels in two consecutive blood samples despite antibiotics; serum blood tests suspicious for PCa; at least one previous negative biopsy; ASAP and/or high-grade PIN; negative digital rectal examination. All eligible patients will undergo 68Ga-PSMA PET/CT and mpMRI scans within 1 month's distance from each other, followed by biopsy session to be completed within 1 month's distance. Targeted TRUS fusion needle biopsy will be performed for all lesions detected with PET and mpMRI. The total duration of the study is 36 months. KEY MESSAGES: By comparing the "experimental test" and the "standard test" in parallel, we will be able to determine the superior diagnostic performance of 68Ga-PSMA PET/CT over mpMRI in detecting PCa, and in particular clinically significant PCa, in the specific cohort of patients with a high suspicion of PCa who are candidates to re-biopsy. The clinical impact of the "experimental test" will be subsequently analyzed in terms of the number of prostate biopsies that could be spared, time-consuming, patient friendliness, and cost-effectiveness.

Exploring the CXCR4/CXCR7/CXCL12 Axis in Primary Desmoid Tumors.

BACKGROUND: Desmoid tumors have an extremely variable natural history. The uncertainty behind desmoid behavior reflects the complexity, which subtends its development and non-linear advancement. Apart from Wnt- ßcatenin mutation, estrogen receptors, and COX-2 overexpression, little is known about the ability of desmoids to grow and recur while being unable to metastasize. Several tumors have been shown to express the CXCR4/CXCR7/CXCL12 axis, whose functions are essential for tumoral development. AIMS: This study aimed to investigate the expression of the CXCR4/CXCR7/CXCL12 axis in primary desmoid tumors and discuss the potential role of this key-signaling as an antiangiogenic therapeutic strategy. METHODS: In this study, 3 µm-thick consecutive sections from each formalin-fixed and paraffin-embedded tissue block were treated with mouse monoclonal antibodies developed against CD34, CXCR4, CXCR7, and CXCL12. RESULTS: Two distinct vessel populations: CXCR4+ and CXCR4- vessels, have been found. Similarly, chemokine receptor CXCR7 expression in the entire desmoid tumor series positively stained a portion of tumor-associated vessels, identifying two distinct subpopulations of vessels: CXCR7+ and CXCR7- vessels. All 8 neoplastic tissue samples expressed CXCL12. Immunohistochemical positivity was identified in both stromal and endothelial vascular cells. Compared to CXCR4 and CXCR7, the vast majority of tumor-associated vessels were found to express this chemokine. CONCLUSION: It is the first time, as per our knowledge, that CXCR4/CXCR7/CXCL12 axis expression has been identified in a desmoid type-fibromatosis series. CXCL12 expression by neoplastic cells, together with CXCR4 and CXCR7 expression by a subgroup of tumor-associated vessels, was detected in all desmoid tumor tissue samples examined. Since chemokines are known contributors to neovascularization, CXCR4/CXCR7/CXCL12 axis may play a role in angiogenesis in this soft-tissue tumor histotype, thereby supporting its growth.

Prospective Validation of the ROL System in Substaging pT1 High-Grade Urothelial Carcinoma: Results from a Mono-Institutional Confirmatory Analysis in BCG Treated Patients.

Patients with pT1 high-grade (HG) urothelial carcinoma (UC) and a very high risk of progression might benefit from immediate radical cystectomy (RC), but this option remains controversial. Validation of a standardized method to evaluate the extent of lamina propria (LP) invasion (with recognized prognostic value) in transurethral resection (TURBT) specimens is still needed. The Rete Oncologica Lombarda (ROL) system showed a high predictive value for progression after TURBT in recent retrospective studies. The ROL system was supposed to be validated on a large prospective series of primary urothelial carcinomas from a single institution. From 2016 to 2020, we adopted ROL for all patients with pT1 HG UC on TURBT. We employed a 1.0-mm threshold to stratify tumors in ROL1 and ROL2. A total of 222 pT1 HG UC were analyzed. The median age was 74 years, with a predominance of men (73.8%). ROL was feasible in all cases: 91 cases were ROL1 (41%), and 131 were ROL2 (59%). At a median follow-up of 26.9 months (IQR 13.8-40.6), we registered 81 recurrences and 40 progressions. ROL was a significant predictor of tumor progression in both univariable (HR 3.53; CI 95% 1.56-7.99; p < 0.01) and multivariable (HR 2.88; CI 95% 1.24-6.66; p = 0.01) Cox regression analyses. At Kaplan-Meier estimates, ROL showed a correlation with both PFS (p = 0.0012) and RFS (p = 0.0167). Our results confirmed the strong predictive value of ROL for progression in a large prospective series. We encourage the application of ROL for reporting the extent of LP invasion, substaging T1 HG UC, and improving risk tables for urological decision-making.

Non-muscle invasive bladder cancer biomarkers beyond morphology.

Non-muscle invasive bladder cancer (NMIBC) still represents a challenge in decision-making and clinical management since prognostic and predictive biomarkers of response to treatment are still under investigation. In addition to the risk factors defined by EORTC guidelines, histological features have also been considered key variables able to impact on recurrence and progression in bladder cancer. Conversely, the role of genomic rearrangements or expression of specific proteins at tissue level need further assessment in NMIBC. As with muscle-invasive cancer, NMIBC is a heterogeneous disease, characterized by genomic instability, varying rates of mutation and a wide range of protein tissue expression. In this Review, we summarized the recent evidence on prognostic and predictive tissue biomarkers in NMIBC, beyond morphological parameters, outlining how they could affect tumor biology and consequently its behavior during clinical care. Our aim was to facilitate clinical evaluation of promising biomarkers that may be employed to better stratify patients. We described the most common molecular events and immunohistochemical protein expressions linked to recurrence and progression. Moreover, we discussed the link between available treatments and molecular drivers that could be predictive of clinical response. In conclusion, we foster further investigations with particular focus on immunohistochemical evaluation of tissue biomarkers, a promising and cost-effective tool for daily practice.

Stereotactic Body Radiation Therapy for Lung Metastases From Sarcoma in Oligometastatic Patients: A Phase 2 Study.

PURPOSE: The lung is the most frequent site of metastasis in patients with sarcoma. Pulmonary metastasectomy is the most common treatment performed. Stereotactic body radiation therapy (SBRT) has proven to be a potential alternative to resection. This prospective phase 2 study aimed to assess the role of SBRT for patients with lung metastases. METHODS AND MATERIALS: Adult patients with up to 4 lung metastases (LMs) ≤5 cm in diameter and unsuitable for surgery were included. Dose prescription was based on site and size: 30 Gy/1 fraction for peripheral lesions ≤10 mm, 60 Gy/3 fractions for peripheral lesions 11 to 20 mm, 48 Gy/4 fractions for peripheral lesions >20 mm, and 60 Gy/8 fractions for central lesions. The primary endpoint was the proportion of treated lesions free from progression at 12 months. Secondary endpoints were disease-free survival (DFS), overall survival (OS), and toxicity. RESULTS: Between March 2015 and December 2020, 44 patients with a total of 71 LMs were enrolled. Twelve-month local control was 98.5% ± 1.4%, reaching the primary aim; the median DFS time was 12 months (95% CI, 8-16 months), and the 1-, 2-, 3-, 4-, and 5-year PFS rates were 50% ± 7.5%, 19.5% ± 6.6%, 11.7% ± 5.8%, 11.7% ± 5.8%, and 11.7% ± 5.8%, respectively. The median OS time was 49 months (95% CI, 24-49 months), and the 1-, 2-, 3-, 4-, and 5-year OS rates were 88.6% ± 4.7%, 66.7 ± 7.6%, 56.8% ± 8.4%, 53.0% ± 8.6%, and 48.2% ± 9.1%, respectively. Prognostic factors recorded as significantly affecting survival were age, grade of primary sarcoma, interval time from diagnosis to occurrence of LMs, and number of LMs. No severe pulmonary toxicity (grade 3-4) occurred. CONCLUSIONS: The study found a local control of LMs in almost all patients treated, with negligible toxicity. Survival was also highly satisfactory. Well-designed randomized trials comparing surgery with SBRT for patients with metastatic lung sarcoma are needed to confirm these preliminary data.

Intratumoral Switch of Molecular Phenotype and Overall Survival in Muscle Invasive Bladder Cancer.

In recent years, immunohistochemical protein expression was studied as a surrogate to the molecular classification of bladder cancer, although no tissue biomarkers are available for clinical use to predict survival or the response to neoadjuvant chemotherapy (CT) in UC, as the literature produced conflicting results. This retrospective study included TURB specimens harboring foci of HG pT2 muscle-invasive bladder carcinoma (MIBC) from 251 patients who subsequently underwent radical cystectomy. We performed immunohistochemical analysis on tumor samples, for relevant gene-expression-based markers for basal type (CD44, CK5/6) and luminal type (CK20 and pPARγ). Piescore, investigated in both non-muscle-invasive (NMI) and muscle-invasive (MI) components of the tumor, divided basal and luminal UC-types when at least three of the four markers were consistent with a specific phenotype, mixed types if one/two luminal and basal markers were present simultaneously, and neu-like types when all four markers investigated were negative. Eighteen selected cases were also investigated with RT-PCR to validate, and to increase the specificity of, the immunohistochemical results. We observe an immunophenotypical difference in the NMI and MI components in 96/251 UC patients (38.25%): half of tumors (44/96 cases) have a transition to basal, 36.46% (35/96 cases) to neu-like, 12.5% (12/96 cases) to mixed, and 5.2% (5/96 cases) to luminal phenotypes. Mixed tumors in the NMI component are more likely to change phenotype than other groups, particularly compared with basal tumors, which demonstrate greater stability (only 8/96 cases, p < 0.00001). The transition of luminal tumors to basal display a better OS compared with the transition toward neu-like tumors (p = 0.027). Overall, the phenotypical switch does not affect lymphovascular invasion, pT, DFS, or OS compared with non-switched cases. In the MI component, the presence of CD44 expression, irrespective of score-related phenotype, shows a protective effect in papillary-type UC (OS p = 0.008, HR 0.453, PFS p = 0.07, HR 0.599), and in UC naïve for CT (p = 0.0479). Piescore immunophenotyping reveals an intratumoral phenotypical transition between the NMI and MI components of the same tumor. The molecular change is a common event in the mixed and luminal categories, but not in basal tumors, which show better phenotypical stability. This phenomenon could partially explain the sensitivity of a subset of luminal UC to chemotherapy: good responders could be "non-real" luminal UC, which acquire nasal markers, such as CD44.

Could We Safely Avoid a Second Resection in Selected Patients With T1 Non-Muscle-Invasive Bladder Cancer? Preliminary Results of Cost-Effectiveness Study From HUmanitas New Indications for ReTUR (HuNIRe) Multicenter Prospective Trial.

Objectives: The aim of this study is to assess whether restaging transurethral resection (ReTUR) could be safely replaced with urine cytology (UC) and in-office fiexible cystoscopy in selected T1 non-muscle-invasive bladder cancer (NMIBC). Materials and Methods: This is an ongoing prospective multicenter trial enrolling patients diagnosed with T1 BC from 5 Italian centers. Patients with a macroscopically incomplete initial resection or absence of detrusor muscle were subjected to ReTUR according to European Association of Urology (EAU) guidelines. Conversely, those with a complete tumor resection at initial TUR underwent UC at 3-4 weeks and in-office fiexible white-light and narrow-band cystoscopy at 4-6 weeks. In case of positive UC, or evidence of recurrence at cystoscopy, ReTUR was performed within 2 weeks. Otherwise, patients started Bacillus Calmette-Guérin (BCG) induction course without ReTUR. The primary endpoint was to determine the feasibility and the clinical utility of not performing ReTUR in selected T1 NMIBC patients. The secondary endpoint was to perform a cost-benefit analysis of this alternative approach. Results: Since May 2020, among 87 patients presenting with T1, 76 patients were enrolled. Nineteen (25%) patients underwent standard ReTUR after initial resection, 10 (13.2%) due to the absence of the detrusor muscle and 9 (11.8%) due to a macroscopically incomplete initial TUR. Overall, 57 (75%) patients initially avoided immediate ReTUR and underwent UC plus in-office flexible cystoscopy. Among them, 38 (66.7%) had no evidence of residual disease and immediately started the BCG induction course. Nineteen patients (33.3%) underwent "salvage" ReTUR due to either positive UC (7; 12.3%) or suspicious cystoscopy (12; 21%). Considering only the patients who initially avoided the ReTUR, disease recurrence was observed in 10/57. The saving of resource for each safely avoided ReTUR was estimated to be 1,759 €. Considering the entire sample, we estimated a saving of 855 € per patient if compared with the EAU guideline approach. Conclusion: The preliminary results of our trial suggested that ReTUR might be safely avoided in highly selected T1 BC patients with a complete resection at first TUR. Longer follow-up and larger sample size are needed to investigate the long-term oncological outcomes of this alternative approach.

Validation of a Novel Three-Dimensional (3D Fusion) Gross Sampling Protocol for Clear Cell Renal Cell Carcinoma to Overcome Intratumoral Heterogeneity: The Meet-Uro 18 Study.

We aimed to overcome intratumoral heterogeneity in clear cell renal cell carcinoma (clearRCC). One hundred cases of clearRCC were sampled. First, usual standard sampling was applied (1 block/cm of tumor); second, the whole tumor was sampled, and 0.6 mm cores were taken from each block to construct a tissue microarray; third, the residual tissue, mapped by taking pieces 0.5 × 0.5 cm, reconstructed the entire tumor mass. Precisely, six randomly derived pieces of tissues were placed in each cassette, with the number of cassettes being based on the diameter of the tumor (called multisite 3D fusion). Angiogenic and immune markers were tested. Routine 5231 tissue blocks were obtained. Multisite 3D fusion sections showed pattern A, homogeneous high vascular density (10%), pattern B, homogeneous low vascular density (8%) and pattern C, heterogeneous angiogenic signatures (82%). PD-L1 expression was seen as diffuse (7%), low (33%) and absent (60%). Tumor-infiltrating CD8 scored high in 25% (pattern hot), low in 65% (pattern weak) and zero in 10% of cases (pattern desert). Grading was upgraded in 26% of cases (G3-G4), necrosis and sarcomatoid/rhabdoid characters were observed in, respectively, 11 and 7% of cases after 3D fusion (p = 0.03). CD8 and PD-L1 immune expressions were higher in the undifferentiated G4/rhabdoid/sarcomatoid clearRCC subtypes (p = 0.03). Again, 22% of cases were set to intermediate to high risk of clinical recurrence due to new morphological findings of all aggressive G4, sarcomatoid/rhabdoid features by using 3D fusion compared to standard methods (p = 0.04). In conclusion, we propose an easy-to-apply multisite 3D fusion sampling that negates bias due to tumor heterogeneity.

Xpert Bladder Cancer Monitor May Avoid Cystoscopies in Patients Under "Active Surveillance" for Recurrent Bladder Cancer (BIAS Project): Longitudinal Cohort Study.

OBJECTIVES: To test the hypothesis that patients under active surveillance (AS) for Non-muscle Invasive Bladder Cancer (NMIBC) who were negative on longitudinal re-testing by the Xpert® Bladder Cancer Monitor (Xpert BC Monitor) assay may avoid unnecessary cystoscopies and urine cytology (UC). SUBJECTS/PATIENTS OR MATERIALS AND METHODS: This is a prospective cohort study of patients enrolled in the AS protocol for recurrent NMIBC (Bladder Cancer Italian Active Surveillance, BIAS project), whose urine samples were analyzed by Xpert BC Monitor upon entry in the study (T0). Patients who had a negative Xpert test and did not fail AS, underwent additional Xpert tests after 4 (T1), 8 (T2), and 12 (T3) months. The clinical utility of Xpert was assessed by determining the number of cystoscopies and UC that could be avoided within 1 year. RESULTS: Overall, 139 patients were tested with Xpert at T0. Median follow-up was 23 (IQR 17-27) months. Sixty-eight (48.9%) patients failed AS, 65 (46.7%) are currently on AS, and 6 (4.3%) were lost at follow-up. At T0 57 (41.0%) patients had a negative test and 36 (63.2%) are still in AS. In patients with 2 consecutives negative Xpert tests, we could have avoided 73.9% of unnecessary cystoscopies, missing 26.4% failure, up to avoid all cystoscopies with 4 negative tests missing only 12% of failure. All the patients with negative Xpert had negative UC. Failure-free-survival at median follow-up (23 month) stratified for having 0, 1, or ≥2 negative tests was 67.0, 55.1. and 84.1, respectively. CONCLUSION: Our findings suggest that Xpert BC Monitor assay, when it is longitudinally repeated, could significantly reduce the number of unnecessary cystoscopies and UC during their follow-up.

Case Report: Unclassified Renal Cell Carcinoma With Medullary Phenotype and SMARCB1/INI1 Deficiency, Broadening the Spectrum of Medullary Carcinoma.

Renal medullary carcinoma (RMC) is a rare entity with poor prognosis bearing inactivating genomic alterations in SMARCB1/INI1 resulting in the loss of expression of INI1 and occurring in young patients with sickle cell trait or sickle cell disease. Recently, rare examples with histological characteristics of RMC have been described in older patients without hemoglobinopathies and provisionally termed "Renal cell carcinoma unclassified with medullary phenotype" (RCCU-MP). Fluorescence in situ Hybridization (FISH) can detect alterations in SMARCB1/INI1 consisting mostly in inactivating translocation of one allele and deletion of the second. To date, only seven further cases of RCCU-MP have been described in the literature. Here we report the second Italian case of RCCU-MP, a 62-year-old man presenting with persistent dull back pain and incidentally discovering a 13 cm mass in the right kidney. The nomenclature of this entity is still debated and might be updated as a variant of medullary carcinoma in the upcoming WHO classification. In the meantime, we encourage awareness of these extraordinarily rare neoplasms with poor outcomes.