Blockade of the CD47/SIRPα checkpoint axis potentiates the macrophage-mediated anti-tumor efficacy of tafasitamab.

Macrophages are one of the key mediators of the therapeutic effects exerted by monoclonal antibodies, such as the anti-CD19 antibody tafasitamab, approved in combination with lenalidomide for the treatment of relapsed or refractory (r/r) diffuse large B cell lymphoma (DLBCL). However, antibody-dependent cellular phagocytosis (ADCP) in the tumor microenvironment can be counteracted by increased expression of the inhibitory receptor SIRPα on macrophages and its ligand, the immune checkpoint molecule CD47 on tumor cells. The aim of this study was to investigate the impact of the CD47-SIRPα axis on tafasitamabmediated phagocytosis and explore the potential of anti-CD47 blockade to enhance its antitumor activity. Elevated expression of both SIRPα and CD47 was observed in DLBCL patient-derived lymph node biopsies compared to healthy controls. CRISPR-mediated CD47 overexpression impacted tafasitamab-mediated ADCP in vitro and increased expression of SIRPα on macrophages correlated with decreased ADCP activity of tafasitamab against DLBCL cell lines. Combination of tafasitamab and an anti-CD47 blocking antibody enhanced ADCP activity of in vitro generated macrophages. Importantly, tafasitamab-mediated phagocytosis was elevated in combination with CD47 blockade using primary DLBCL cells and patient-derived lymphoma-associated macrophages (LAMs) in an autologous setting. Furthermore, lymphoma cells with low CD19 expression were efficiently eliminated by the combination treatment. Finally, combined treatment of tafasitamab and an anti-CD47 antibody resulted in enhanced tumor volume reduction and survival benefit in lymphoma xenograft mouse models. These findings provide evidence that CD47 blockade can enhance the phagocytic potential of tumor targeting immunotherapies such as tafasitamab and suggest there is value in exploring the combination in the clinic.

Prognostic and Predictive Potential of CCL5 Expression in Muscle-Invasive Bladder Cancer Patients.

Bladder cancer (BC) is the 12th most commonly diagnosed cancer worldwide. Although there are several well-established molecular and immunological classifications, prognostic and predictive markers for tumor cells and immune cells are still needed. Using a tissue microarray, we analyzed the expression of the chemokine CC motif ligand 5 (CCL5) by immunohistochemistry (IHC) in 175 muscle-invasive BC samples. The application of a single cutoff for the staining status of tumor cells (TCs; positive vs. negative) and immune cells (ICs; positive vs. negative) revealed 75 patients (42.9%) and 123 patients (70.3%) with CCL5-positive TCs or ICs, respectively. IHC results were associated with prognostic and predictive data. Multivariate Cox regression analysis revealed that positive CCL5 staining in TCs was associated with significantly shorter disease-specific survival (DSS; RR = 1.51; p = 0.047), but CCL5-negative ICs were associated with significantly shorter overall survival (OS; RR = 1.66; p = 0.005), DSS (RR = 2.02; p = 0.001) and recurrence-free survival (RFS; RR = 1.94; p = 0.002). Adjuvant chemotherapy was favorable for patients with CCL5-negative ICs for OS (RR = 0.30; p = 0.006), DSS (RR = 0.36; p = 0.022) and RFS (RR = 0.41; p = 0.046) but not for patients with CCL5-positive ICs, except in the subgroup of N1 + N2 patients, where it was associated with better OS. We suggest that CCL5 expression can be a prognostic and predictive marker for muscle-invasive bladder cancer patients.

Prognostic impact of variant histologies in urothelial bladder cancer treated with radical cystectomy.

OBJECTIVES: To evaluate variant histologies (VHs) for disease-specific survival (DSS) in patients with invasive urothelial bladder cancer (BCa) undergoing radical cystectomy (RC). MATERIALS AND METHODS: We analysed a multi-institutional cohort of 1082 patients treated with upfront RC for cT1-4aN0M0 urothelial BCa at eight centres. Univariable and multivariable Cox' regression analyses were used to assess the effect of different VHs on DSS in overall cohort and three stage-based analyses. The stages were defined as 'organ-confined' (≤pT2N0), 'locally advanced' (pT3-4N0) and 'node-positive' (pTanyN1-3). RESULTS: Overall, 784 patients (72.5%) had pure urothelial carcinoma (UC), while the remaining 298 (27.5%) harboured a VH. Squamous differentiation was the most common VH, observed in 166 patients (15.3%), followed by micropapillary (40 patients [3.7%]), sarcomatoid (29 patients [2.7%]), glandular (18 patients [1.7%]), lymphoepithelioma-like (14 patients [1.3%]), small-cell (13 patients [1.2%]), clear-cell (eight patients [0.7%]), nested (seven patients [0.6%]) and plasmacytoid VH (three patients [0.3%]). The median follow-up was 2.3 years. Overall, 534 (49.4%) disease-related deaths occurred. In uni- and multivariable analyses, plasmacytoid and small-cell VHs were associated with worse DSS in the overall cohort (both P = 0.04). In univariable analyses, sarcomatoid VH was significantly associated with worse DSS, while lymphoepithelioma-like VH had favourable DSS compared to pure UC. Clear-cell (P = 0.015) and small-cell (P = 0.011) VH were associated with worse DSS in the organ-confined and node-positive cohorts, respectively. CONCLUSIONS: More than 25% of patients harboured a VH at time of RC. Compared to pure UC, clear-cell, plasmacytoid, small-cell and sarcomatoid VHs were associated with worse DSS, while lymphoepithelioma-like VH was characterized by a DSS benefit. Accurate pathological diagnosis of VHs may ensure tailored counselling to identify patients who require more intensive management.

Prognostic impact of molecular muscle-invasive bladder cancer subtyping approaches and correlations with variant histology in a population-based mono-institutional cystectomy cohort.

PURPOSE: Recently discovered molecular classifications for urothelial bladder cancer appeared to be promising prognostic and predictive biomarkers. The present study was conducted to evaluate the prognostic impact of molecular subtypes assessed by two different methodologies (gene and protein expression), to compare these two approaches and to correlate molecular with histological subtypes in a consecutively collected, mono-institutional muscle-invasive bladder cancer (MIBC) cohort. METHODS: 193 MIBC were pathologically re-evaluated and molecular subtypes were assessed on mRNA (NanoString technology, modified 21-gene-containing MDACC approach) and protein levels (immuno-histochemical [IHC] analysis of CK5, CK14, CD44, CK20, GATA3 and FOXA1). Descriptive statistical methods and uni-/multi-variable survival models were employed to analyze derived data. RESULTS: Neither gene expression nor protein-based subtyping showed significant associations with disease-specific (DSS) or recurrence-free survival (RFS). Agreement between mRNA (reference) and protein-based subtyping amounted 68.6% for basal, 76.1% for luminal and 50.0% for double-negative tumors. Histological subtypes associated with RFS in uni-variable (P = 0.03), but not in multivariable survival analyses. Tumors with variant histology predominantly showed luminal subtypes (gene expression subtyping: 36/55 cases, 65.5%; protein subtyping: 44/55 cases, 80.0%). Squamous differentiation significantly associated with basal subtypes (gene expression subtyping: 44/45 squamous cases, 97.8%; protein subtyping: 36/45 cases, 80.0%). CONCLUSION: In our consecutive cystectomy cohort, neither gene, protein expression-based subtyping, nor histological subtypes associated with DSS or RFS in multi-variably adjusted survival analyses. Application of a limited IHC subtyping marker panel showed high concordance of 83.9% with gene expression-based subtyping, thus underlining the utility for subtyping in pathological routine diagnostics. In addition, histological MIBC subtypes are strong indicators for intrinsic subtypes.

Programmed death ligand 1 (PD-L1) in colon cancer and its interaction with budding and tumor-infiltrating lymphocytes (TILs) as tumor-host antagonists.

PURPOSE: To analyze the role of programmed death ligand 1 (PD-L1) immunohistochemisty in the context of tumor microenvironment in colon cancer (CC) with focus on the interaction between tumor budding and tumor-infiltrating lymphocytes (TILs) and to elucidate its potential value for immunooncologic treatment decisions. METHODS: Three hundred forty seven patients with CC, stages I to IV, were enrolled. PD-L1 immunohistochemistry was performed using two different antibodies (clone 22C3 pharmDx, Agilent and clone QR1, Quartett). Tumor proportion score (TPS) as well as immune cell score (IC) was assessed. Budding and TILs were assessed according to the criteria of the International Tumor Budding Consensus Conference (ITBCC) and International TILs Working Group (ITWG). Correlation analyses as well as survival analyses were performed. RESULTS: PD-L1 positivity significantly correlated with TILs > 5% and MMR deficiency, and PD-L1-positive cases (overall and IC) showed significantly longer overall survival (OS) with both antibodies.The parameters "high grade," "right-sidedness," and "TILS > 5% regardless of MMR status" evolved as potential parameters for additional immunological treatment decisions. Additionally, TPS positivity correlated with low budding. More PD-L1-positive cases were seen in both high TIL groups. The low budding/high TIL group showed longer disease-free survival and longer OS in PD-L1-positive cases. CONCLUSION: Overall, PD-L1 positivity correlated with markers of good prognosis. PD-L1 immunohistochemistry was able to identify parameters as additional potential candidates for immune therapy. Furthermore, it was able to stratify patients within the low budding/high TIL group with significant prognostic impact.

Variant morphology and random chromosomal integration of BK polyomavirus in posttransplant urothelial carcinomas.

BK polyomavirus (BKPyV) causes major complications in solid organ transplant recipients but little is known about its role in the development of urothelial carcinoma (UC) during immunosuppression. Immunohistochemistry (IHC) screening for polyomavirus large T antigen (LTag) was performed in 94 micropapillary UC (MPUC), 480 unselected UC, 199 muscle invasive UC (including 83 UC with variant differentiation), 76 cases of plasmocytoid, nested and large nested UC and 15 posttransplant UC. LTag expressing UC were reevaluated regarding their histomorphological features and characterized by IHC for p53 and HER2, chromogenic in situ hybridization for HER2 and SNaPshot analysis of the TERT promoter and HRAS. Real-time PCR and next generation sequencing (NGS) were performed to search for BKPyV-DNA and for variants in the tumor and viral genomes. We detected five LTag expressing UC which were diagnosed between 2 and 18 years after kidney (n = 4) or heart (n = 1) transplantation. 89 MPUC without history of organ transplantation and overall 755 UC (including cases with variant histology) were LTag negative. Of the five LTag expressing UC, three were MPUC, one showed extensive divergent differentiation with Mullerian type clear cell carcinoma, and one displayed focal villoglandular differentiation. All five tumors had aberrant nuclear p53 expression, 2/5 were HER2-amplified, and 3/5 had TERT promoter mutations. Within the 50 most common cancer related genes altered in UC we detected very few alterations and no TP53 mutations. BKPyV-DNA was present in 5/5 UC, chromosomal integration of the BKPyV genome was detectable in 4/5 UC. Two UC with BKPyV integration showed small deletions in the BKPyV noncoding control region (NCCR). The only UC without detectable BKPyV integration had a high viral load of human herpesvirus 6 (HHV-6). Our results suggest that LTag expression of integrated BKPyV genomes and resulting p53 inactivation lead to aggressive high-grade UC with unusual, often micropapillary morphology.

TERT promoter mutation analysis as a surrogate to morphology and immunohistochemistry in problematic spindle cell lesions of the urinary bladder.

AIMS: Pseudosarcomatous myofibroblastic proliferations (PSMPs) of the urinary bladder are diagnostically challenging. Diagnostic difficulties are mainly due to frequent cytokeratin expression, variable ALK expression and worrisome morphological features suggestive of malignancy. Conversely, sarcomatoid urothelial carcinoma (UC) may show bland inflammatory myofibroblastic tumour (IMT)-like morphology. TERT promoter mutations are characteristic events in urothelial cancers, but have not been studied in PSMPs. METHODS AND RESULTS: We compared histomorphological and immunohistochemical features and TERT promoter status in 16 PSMPs and 18 sarcomatoid UC. In a subset of PSMPs, RNA sequencing was performed. At least focal IMT-like morphology was seen in nine of 17 sarcomatoid UC. Atypical mitoses, differentiated urothelial component and heterologous elements were the most reliable distinguishing histomorphological features of sarcomatoid UC, if present. A panel of immunohistochemistry (IHC) including ALK (clone D5F3), p53 pattern, p63 and GATA3 reliably distinguished PSMP from sarcomatoid UC. GATA3 (P = 0.001) and p53 patterns (mutant versus wild-type; P < 0.001) were differentially expressed between PSMPs and sarcomatoid UC. Diffuse pancytokeratin staining was significantly associated with PSMPs (10 of 13) compared to four of 14 sarcomatoid UCs (P = 0.012). TERT promoter mutations were found in 17 of 18 sarcomatoid UC versus none of 16 PSMPs (P < 0.001). RNA sequencing revealed ALK genetic rearrangements in one of two ALK-positive and one of 10 ALK-negative PSMPs, which revealed a novel FN1/RET gene fusion. CONCLUSION: Careful histomorphological analysis and differential IHC reliably distinguish the majority of PSMPs and sarcomatoid UC. In equivocal cases, TERT promoter mutation analysis and/or detection of ALK expression/rearrangements are valuable additional diagnostic adjuncts, strongly supporting sarcomatoid UC and PSMP, respectively.

Transurethral en bloc submucosal hydrodissection vs conventional resection for resection of non-muscle-invasive bladder cancer (HYBRIDBLUE): a randomised, multicentre trial.

OBJECTIVE: To determine whether transurethral en bloc submucosal hydrodissection of bladder tumours (TUEB) improves the quality of the resection compared to conventional transurethral resection of bladder tumour (TURBT) in patients with non-muscle-invasive bladder cancer (NMIBC). PATIENTS AND METHODS: A randomised, multicentre trial (HYBRIDBLUE) was conducted with a superiority design. Six German academic centres participated between September 2012 and August 2015. Based on literature analysis, a sample size for accurate histopathological assessment concerning muscle invasion was assumed to be feasible in 50% (P0 = 0.5) of TURBT and 80% of TUEB cases. After pre-screening of a total of 305 patients, participants were allocated to two study arms: Group I: hexaminolevulinate (HAL)-guided TUEB; Group II: conventional HAL-guided TURBT. The primary endpoint was the proportion of specimens that could be reliably evaluated pathologically concerning muscle invasiveness. Secondary endpoints included rates of histopathological completeness of the resection, muscularis propria content, recurrence, and complication rates. RESULTS: A total of 115 patients (TUEB 56; TURBT 59) were eligible for final analysis. Adequate histopathological assessment, which included muscularis propria content and tumour margins (R0 vs R1), was present in 48/56 (86%) TUEB patients compared to 37/59 (63%; P = 0.006) in the TURBT group. R0 was confirmed in 30/56 TUEB patients (57%) and five of 59 TURBT patients (9%; P < 0.001). No complications of Grade ≥III were observed in both arms. At 3 and 12 months, three and 19 patients recurred in the TUEB group vs seven and 11 patients in the TURBT group, respectively (P = 0.33 and P = 0.08). CONCLUSIONS: In this randomised study, TUEB was shown to be clinically safe regarding perioperative endpoints. An adequate histopathological assessment concerning muscle invasion was significantly better assessable in the TUEB arm compared to standard TURBT. This finding indicates the clinical potential for reducing the rate of early re-resections. Yet, a larger study with recurrence-free survival as the primary endpoint is needed to assess the oncological efficacy between both techniques.

Isolated thrombotic microangiopathy of the small intestine in a patient with atypical hemolytic uremic syndrome - a case report.

BACKGROUND: Atypical hemolytic uremic syndrome (aHUS) is a rare disease characterized by systemic thrombotic microangiopathy (TMA) reflected by hemolysis, anemia, thrombocytopenia and systemic organ injury. The optimal management of aHUS-patients when undergoing kidney transplantation to prevent recurrence in the allograft is eculizumab, an approved recombinant antibody targeting human complement component C5. CASE PRESENTATION: A 39 year-old woman presented with severe abdominal pain, diarrhea and emesis for 3 days. In her past medical history she had experienced an episode of aHUS leading to end stage renal disease (ESRD) in 2007 and a genetic workup revealed a heterozygous mutation in the membrane cofactor protein gene. In 2014 she underwent cadaveric kidney transplantation. Four years later she had to go back on hemodialysis due to allograft failure following a severe systemic cytomegalovirus infection resulting in transplant failure. At presentation she still received calcineurin-inhibitor therapy and reported subfebrile temperatures and pain projecting over the transplant prior to the current symptoms. A contrast enhanced CT-scan of the abdomen revealed inflammatory wall thickening of the small intestine. Diagnostic endoscopy discovered fresh blood in the small intestine without a clear source of bleeding. Histopathology of the small intestine biopsies showed severe thrombotic microangiopathy. Of note, the patient persistently had no signs of systemic hemolysis. Since the TMA of the small intestine was most likely due to aHUS, eculizumab treatment was initiated which abolished the symptoms. CONCLUSION: Here we report a patient with thrombotic microangiopathy with predominant manifestation in a single organ, the small intestine, due to aHUS with absence of systemic signs and symptoms. aHUS patients usually require a secondary trigger for the disease to manifest. In this case, the trigger may be attributed to the dysfunctional renal transplant, which was subsequently explanted. Histology of the explanted kidney showed severe inflammation due to purulent nephritis and signs of cellular rejection. After nephrectomy, we continued eculizumab therapy until the patient completely recovered. No signs of TMA recurred after discontinuation of eculizumab, further supporting the concept of the renal transplant as the main trigger of TMA of the small intestine in our patient.

High Stroma T-Cell Infiltration is Associated with Better Survival in Stage pT1 Bladder Cancer.

Stage pT1 bladder cancer (BC) shows highly diverse outcomes. Predictive markers are required to stratify patients for personalized treatment. The present study aimed to validate immune response quantification as a prognostic marker. Patients with pT1 BC (n = 167) treated by transurethral resection of the bladder (TURB) were enrolled. Formaldehyde-fixed paraffin-embedded material was stained for CD3 and CD8. Corresponding T cells were counted in three regions with the highest immune response. Numbers of tertiary lymphoid structures (TLS) and lymphocyte aggregates (LA) were quantified. High CD3+ stroma T-cell infiltration was associated with improved survival (p = 0.045), especially in the G3 subgroup (p = 0.01). Cluster with higher immune response showed less recurrence (p = 0.034) and favorable overall survival (OS) (p = 0.019). In contrast, higher CD3+ and CD8+ tumor T-cell infiltration seemed to have a negative impact on prognosis. TLS and LA were more frequently observed in G3 tumors, indicating an increased anti-tumoral immune response. We proved the role of immune cell infiltration and showed that higher infiltration numbers of CD3+ (not CD8+) lymphocytes in the stroma are associated with favorable outcome. Immune cell quantification could be used as a marker to help stratify patients' risk and therefore, to optimize patients' management and follow-up examination as well as possible therapies.

Long-Term Experience of Chemoradiotherapy Combined with Deep Regional Hyperthermia for Organ Preservation in High-Risk Bladder Cancer (Ta, Tis, T1, T2).

BACKGROUND: The aim of this study was to evaluate the efficacy and safety of chemoradiotherapy (RCT) combined with regional deep hyperthermia (RHT) of high-risk bladder cancer after transurethral resection of bladder tumor (TUR-BT). MATERIALS AND METHODS: Between 1982 and 2016, 369 patients with pTa, pTis, pT1, and pT2 cN0-1 cM0 bladder cancer were treated with a multimodal treatment after TUR-BT. All patients received radiotherapy (RT) of the bladder and regional lymph nodes. RCT was administered to 215 patients, RCT + RHT was administered to 79 patients, and RT was used in 75 patients. Treatment response was evaluated 4-6 weeks after treatment with TUR-BT. RESULTS: Complete response (CR) overall was 83% (290/351), and in treatment groups was RT 68% (45/66), RCT 86% (178/208), and RCT + RHT 87% (67/77). CR was significantly improved by concurrent RCT compared with RT (odds ratio [OR], 2.32; 95% confidence interval [CI], 1.05-5.12; p = .037), less influenced by hyperthermia (OR, 2.56; 95% CI, 0.88-8.00; p = .092). Overall survival (OS) after RCT was superior to RT (hazard ratio [HR], 0.7; 95% CI, 0.50-0.99; p = .045). Five-year OS from unadjusted Kaplan-Meier estimates was RCT 64% versus RT 45%. Additional RHT increased 5-year OS to 87% (HR, 0.32; 95% CI, 0.18-0.58; p = .0001). RCT + RHT compared with RCT showed a significantly better bladder-preservation rate (HR, 0.13; 95% CI, 0.03-0.56; p = .006). Median follow-up was 71 months. The median number of RHT sessions was five. CONCLUSION: The multimodal treatment consisted of a maximal TUR-BT followed by RT; concomitant platinum-based chemotherapy combined with RHT in patients with high-grade bladder cancer improves local control, bladder-preservation rate, and OS. It offers a promising alternative to surgical therapies like radical cystectomy. IMPLICATIONS FOR PRACTICE: Radical cystectomy with appropriate lymph node dissection has long represented the standard of care for muscle-invasive bladder cancer in medically fit patients, despite many centers reporting excellent long-term results for bladder preserving strategies. This retrospective analysis compares different therapeutic modalities in bladder-preservation therapy. The results of this study show that multimodal treatment consisting of maximal transurethral resection of bladder tumor followed by radiotherapy, concomitant platinum-based chemotherapy combined with regional deep hyperthermia in patients with Ta, Tis, T1-2 bladder carcinomas improves local control, bladder-preservation rate, and survival. More importantly, these findings offer a promising alternative to surgical therapies like radical cystectomy. The authors hope that, in the future, closer collaboration between urologists and radiotherapists will further improve treatments and therapies for the benefit of patients.

Distinct genetic alterations and luminal molecular subtype in nested variant of urothelial carcinoma.

AIMS: Nested variant of urothelial carcinoma (NVUC) is rare, and only a few small series exist. Molecular characteristics and the classifying marker profile as well as therapeutic targets of this specific variant are mostly unknown. The aim of this study was to characterise NVUC at the molecular level in one of the largest cohorts to date. In addition, we applied an immunohistochemical marker panel in order to define the molecular subtype. METHODS AND RESULTS: Sixty NVUC cases were collected from different departments. TERT promoter mutation analysis was carried out in all samples using SNaPshot analysis. Targeted sequencing of 48 cancer-related genes by next-generation sequencing (NGS) analysis was performed in a subset of 26 cases. Immunohistochemical markers CD44, CK5, CK14, EGFR, p63, FOXA1, GATA3, CD24 and CK20 were used to elucidate the molecular subtype. A total of 62.5% of NVUC cases harboured a mutation of the TERT promoter. Additionally, TP53, JAK3 and CTNNB1 were among the most frequently mutated genes identified by NGS analysis. Subtyping revealed that all NVUC express luminal markers such as CD24, FOXA1, GATA3 and CK20. CONCLUSIONS: In summary, NVUC belong to the luminal molecular subtype. Moreover, a subset of NVUC seems to be characterised by mutations of the Wnt and inflammatory pathways, including JAK3 mutations, indicating a different biological background compared to conventional urothelial bladder cancer.

No Evidence of Microsatellite Instability and Loss of Mismatch-Repair-Protein Expression in Squamous Cell Carcinoma of the Penis.

OBJECTIVE: Microsatellite instability (MSI) and a defective mismatch repair (MMR) system were described as beneficial tumor features for response to immune checkpoint therapy (PD-1 blockade). Meanwhile, the FDA approved PD-1/PD-L1 inhibition treatment for any solid tumor showing MSI and/or defects in the MMR system. For squamous cell carcinoma (SCC) of the penis, no data on the frequency of MSI and altered MMR protein expression are available to date. Therefore, we investigated the MSI status and the expression of MMR proteins in a large cohort of penile SCCs. METHODS: The MSI status of 105 archival formalin-fixed, paraffin-embedded penile SCCs was analyzed using the 5 markers of the NCI consensus panel for MIS testing (BAT25, 26, D2S123, D17S250, and D5S346), or, in cases without representative nontumorous tissue using a validated panel of 5 quasimonomorphic mononucleotide repeat markers (BAT 25, 26 and NR21, 24, 27). The expression of the MMR proteins MLH1, MSH2, MSH6, and PMS2 was analyzed using immunohistochemistry and a tissue microarray of a subset of penile SCCs from our cohort (n = 75). RESULTS: Overall, in 96/105 cases, at least 4 microsatellite markers gave interpretable results. None of the cases showed MSI. Immunohistochemistry for MMR proteins was analyzable in 70/75 cases. All cases showed a regular expression of the MMR proteins. CONCLUSION: MSI and defects in MMR protein expression are not regular features of penile SCC and might not act as biomarkers for PD-1/PD-L1 blockade therapy in penile carcinoma.

The World Health Organization 1973 classification system for grade is an important prognosticator in T1 non-muscle-invasive bladder cancer.

OBJECTIVES: To compare the prognostic value of the World Health Organization (WHO) 1973 and 2004 classification systems for grade in T1 bladder cancer (T1-BC), as both are currently recommended in international guidelines. PATIENTS AND METHODS: Three uro-pathologists re-revised slides of 601 primary (first diagnosis) T1-BCs, initially managed conservatively (bacille Calmette-Guérin) in four hospitals. Grade was defined according to WHO1973 (Grade 1-3) and WHO2004 (low-grade [LG] and high-grade [HG]). This resulted in a lack of Grade 1 tumours, 188 (31%) Grade 2, and 413 (69%) Grade 3 tumours. There were 47 LG (8%) vs 554 (92%) HG tumours. We determined the prognostic value for progression-free survival (PFS) and cancer-specific survival (CSS) in Cox-regression models and corrected for age, sex, multiplicity, size and concomitant carcinoma in situ. RESULTS: At a median follow-up of 5.9 years, 148 patients showed progression and 94 died from BC. The WHO1973 Grade 3 was negatively associated with PFS (hazard ratio [HR] 2.1) and CSS (HR 3.4), whilst WHO2004 grade was not prognostic. On multivariable analysis, WHO1973 grade was the only prognostic factor for progression (HR 2.0). Grade 3 tumours (HR 3.0), older age (HR 1.03) and tumour size >3 cm (HR 1.8) were all independently associated with worse CSS. CONCLUSION: The WHO1973 classification system for grade has strong prognostic value in T1-BC, compared to the WHO2004 system. Our present results suggest that WHO1973 grade cannot be replaced by the WHO2004 classification in non-muscle-invasive BC guidelines.

Infiltrative lamina propria invasion pattern as an independent predictor for cancer-specific and overall survival of instillation treatment-naïve stage T1 high-grade urothelial bladder cancer.

OBJECTIVES: To investigate established prognostic factors and relatively new histopathological tumor characteristics including metric substage and lamina propria invasion patterns in a large series of T1 high-grade non-muscle-invasive bladder cancer. METHODS: Between 1989 and 2012, 322 patients with initial stage T1 high-grade bladder cancer underwent transurethral resection, followed by re-transurethral resection and a conservative approach with follow-up regime alone or instillation treatment. Transurethral resection specimens were reassessed by two experienced urological pathologists for tumor grade according to the World Health Organization 1973 classification, metric T1 substage, lamina propria invasion pattern and associated carcinoma in situ. The median follow-up period was 42 months (interquartile range 25-72 months). In addition to Kaplan-Meier analyses, uni- and multivariable Cox regression analyses were used to compare progression-free survival, cancer-specific survival and overall survival for the studied parameters comparing two subcohorts. RESULTS: While in patients after instillation treatment no examined feature was shown as an independent predictor for prognosis, there were predictive histopathological features in multivariable Cox regression analyses in instillation treatment-naïve patients: associated carcinoma in situ (hazard ratio 2.278, 95% confidence interval 1.119-4.634, P = 0.023) and World Health Organization 1973 grade 3 (hazard ratio 2.950, 95% confidence interval 1.021-8.536, P = 0.046) for worse progression-free survival, infiltrative lamina propria tumor pattern for worse cancer-specific survival (hazard ratio 2.369, 95% confidence interval 1.034-5.429, P = 0.042) and overall survival (hazard ratio 1.049, 95% confidence interval 1.024-1.075, P = 0.001). CONCLUSIONS: The results of the present T1 high-grade bladder cancer series suggest that lamina propria invasion pattern is a promising parameter to predict the prognosis of T1 high-grade bladder cancer in an instillation treatment-naïve subcohort. Prospective multicenter evaluations are warranted. The need for instillation treatment in T1 high-grade bladder cancer is clearly demanded.

Large nested variant of urothelial carcinoma: a clinicopathological study of 36 cases.

AIMS: The large nested variant of urothelial carcinoma (LNUC) has been added to the World Health Organization (WHO) 2016 classification. Scant data exist, and little is known about its clinical behaviour. MATERIAL AND RESULTS: Cases fulfilling the morphological criteria of LNUC were collected. Pure and mixed cases (i.e. with other patterns of invasive UC) were studied. Immunohistochemical staining with cytokeratin (CK)7, p63, GATA-3, CK20, p53 and Ki-67 was performed. Included were 26 cystectomies (RC) and 10 resections (TURB) belonging to 36 patients with an average age of 66.7 years. Fourteen (39%) were pure LNUCs, and 22 (61%) displayed mixed features. Seventy per cent of the TURBs had pT2 tumours, while 58% of RCs had extravesical disease (≥pT3 and/or ≥pN1), with the rate of advanced disease being higher in mixed (69%) in comparison to pure cases (40%). Similarly, 38% of mixed cases had nodal metastases in comparison to 20% of pure cases. Overall, eight patients (24%) died of disease at a mean interval time of 21.7 months and seven patients (21%) showed recurrence or metastases. Disease progression was significantly higher in mixed cases (55 and 31% in mixed and pure cases, respectively). Positive staining was: CK7 = 87.5%, CK20 = 72%, GATA-3 = 91%, P63 = 100%, p53 = 56% and Ki-67 = mean of 16%. CONCLUSION: Despite the bland cytological appearance and deceptive pattern of invasion of the LNUC, our study validates its fully malignant potential with metastatic spread and tumour-related deaths. Distinguishing mixed from pure LNUCs seems to be of value. LNUCs show comparable immunophenotype to both conventional urothelial carcinoma and the nested variant of urothelial carcinoma.

A Multiplex Kindred with Hennekam Syndrome due to Homozygosity for a CCBE1 Mutation that does not Prevent Protein Expression.

Collagen and calcium-binding EGF domain-containing protein 1 (CCBE1) bi-allelic mutations have been associated with syndromes of widespread congenital lymphatic dysplasia, including Hennekam Syndrome (HS). HS is characterized by lymphedema, lymphangiectasia, and intellectual disability. CCBE1 encodes a putative extracellular matrix protein but the HS-causing mutations have not been studied biochemically. We report two HS siblings, born to consanguineous parents of Turkish ancestry, whose clinical phenotype also includes protein losing enteropathy, painful relapsing chylous ascites, and hypogammaglobulinemia. We identified by whole exome and Sanger sequencing the homozygous CCBE1 C174Y mutation in both siblings. This mutation had been previously reported in another HS kindred from the Netherlands. In over-expression studies, we found increased intracellular expression of all forms (monomers, dimers, trimers) of the CCBE1 C174Y mutant protein, by Western blot, despite mutant mRNA levels similar to wild-type (WT). In addition, we detected increased secretion of the mutant CCBE1 protein by ELISA. We further found the mutant and WT proteins to be evenly distributed in the cytoplasm, by immunofluorescence and confocal microscopy. Finally, we found a strong decrease of lymphatic vessels, with a corresponding diminished expression of CCBE1, by immunohistochemistry of the patients' intestinal biopsies. In contrast, mucosal blood vessels and muscularis mucosae showed normal CCBE1 staining. Our findings show that the mutant CCBE1 C174Y protein is not loss-of-function by loss-of-expression.

Piwil 2 expression is correlated with disease-specific and progression-free survival of chemotherapy-treated bladder cancer patients.

Piwi-like 2 (Piwil 2) belongs to the family of Argonaute genes/proteins. The expression of Piwil 2 is associated with stem cells. A role in tumorigenesis and/or tumor progression is proposed for different cancers but not yet for bladder cancer (BCa). We investigated the Piwil 2 expression by immunohistochemistry in a cohort of 202 BCa patients treated by cystectomy and adjuvant chemotherapy. The association between Piwil 2 expression and disease-specific (DSS) or progression-free survival (PFS) was calculated using Kaplan Meier analyses and univariate/multivariate Cox's regression hazard models.In a multivariate Cox's regression, Piwil 2 expression, either in the cytoplasm or the nucleus, was significantly associated with DSS and PFS. A weak cytoplasmic staining pattern was associated with poor DSS and tumor progression (RR=2.7; P=0.004 and RR=2.4; P=0.027). Likewise,, absent nuclear Piwil 2 immunoreactivity was associated with poor DSS and tumor progression (RR=2.3; P=0.023 and RR=2.2; P=0.022). BCa patients whose tumors exhibited a combination of weak cytoplasmic and absent nuclear immunoreactivity had a 6-fold increased risk of tumor-related death (P=0.005) compared to patients with strong expression. Considering only patients with high grade G3 tumors, a 7.8-fold risk of tumor-associated death and a 3.6-fold risk of tumor progression were detected independently of the histologic tumor subtype or the chemotherapy regimen. In summary, a combination of weak cytoplasmic and absent nuclear expression of Piwil 2 is significantly associated with an increased risk of DSS and tumor progression. This implicates that Piwil 2 could be a valuable prognostic marker for high-risk BCa patients.

Epigenetic inactivation of ITIH5 promotes bladder cancer progression and predicts early relapse of pT1 high-grade urothelial tumours.

Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) has been associated with tumour suppression in various cancers. However, its putative role in bladder cancer is completely unknown. Therefore, we initiated a study analysing ITIH5 expression as well as its prognostic and functional impact on human urothelial cancers (UCs). Expression analysis showed a clear down-regulation of ITIH5 mRNA in 61% (n = 45) of UCs, especially in muscle-invasive tumours (P < 0.001). ITIH5 loss in UCs was further evident on protein level (65.5%, n = 55) as detected by immunohistochemistry. DNA methylation analysis demonstrated tumour-specific ITIH5 promoter methylation in 50% of papillary none-invasive pTa (n = 30) and 68% of invasive (n = 28) UCs. Aberrant ITIH5 promoter methylation in bladder tumours was tightly linked (P < 0.001) with loss of ITIH5 mRNA expression, which was furthermore functionally confirmed by demethylation analysis in cell lines. Pyrosequencing analysis revealed that ITIH5 promoter hypermethylation was closely associated with progressive bladder cancers. Subsequently, a large cohort (n = 120) of clinically challenging pT1 high-grade UC was analysed for ITIH5 expression. Of clinical significance, we found an association between loss of ITIH5 expression and unfavourable prognosis of UC patients without distant metastasis at first diagnosis (recurrence-free survival; hazard ratio: 4.35, P = 0.048). Functionally, ITIH5 re-expression in human RT112 bladder cancer cells led to both suppression of cell migration and inhibition of colony spreading. Hence, we provide evidence that down-regulation of ITIH5 by aberrant DNA hypermethylation may provoke invasive phenotypes in human bladder cancer. Moreover, ITIH5 protein might become a prognostic biomarker for relapse risk stratification in high-grade UC patients.

Proposal for a Novel Histological Scoring System as a Potential Grading Approach for Muscle-invasive Urothelial Bladder Cancer Correlating with Disease Aggressiveness and Patient Outcomes.

BACKGROUND: Grading of muscle-invasive bladder cancer (MIBC) according to the current World Health Organization (WHO) criteria is controversial due to its limited prognostic value. All MIBC cases except a tiny minority are of high grade. OBJECTIVE: To develop a prognostic histological scoring system for MIBC integrating histomorphological phenotype, stromal tumor-infiltrating lymphocytes (sTILs), tumor budding, and growth and spreading patterns. DESIGN, SETTING, AND PARTICIPANTS: Tissue specimens and clinical data of 484 patients receiving cystectomy and lymphadenectomy with curative intent with or without adjuvant chemotherapy. Histomorphological phenotypes, sTILs, tumor budding, and growth and spreading patterns were evaluated and categorized into four grade groups (GGs). GGs were correlated with molecular subtypes, immune infiltration, immune checkpoint expression, extracellular matrix (ECM) remodeling, and epithelial-mesenchymal transition (EMT) activity. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: GGs were associated with overall (OS), disease-specific (DSS), and progression-free (PFS) survival in univariable and multivariable analyses. Association with biological features was analyzed with descriptive statistics. RESULTS AND LIMITATIONS: Integration of two histomorphological tumor groups, three sTILs groups, three tumor budding groups, and four growth/spread patterns yielded four novel GGs that had high significance in the univariable survival analysis (OS, DSS, and PFS). GGs were confirmed as independent prognostic predictors with the greatest effect in the multivariable Cox regression analysis. Correlation with molecular data showed a gradual transition from basal to luminal subtypes from GG1 to GG4; a gradual decrease in survival, immune infiltration, and immune checkpoint activity; and a gradual increase in ECM remodeling and EMT activity. CONCLUSIONS: We propose a novel, prognostically relevant, and biologically based scoring system for MIBC in cystectomies applicable to routine pathological sections. PATIENT SUMMARY: We developed a novel approach to assess the aggressiveness of advanced bladder cancer, which allows improved risk stratification compared with the method currently proposed by the World Health Organization.