Frontline immune checkpoint inhibitors in patients ≥ 90 years with advanced urothelial cancer: a single center experience.

INTRODUCTION: Immune checkpoint inhibitors (ICIs) are approved for advanced urothelial cancer alone and as first-line in combination with enfortumab vedotin. Platinum based chemotherapy which is another frontline choice is often not a treatment option for older patients due to comorbidities that increase with age. Despite ICIs being better tolerated compared to traditional chemotherapy little is known about their efficacy and toxicity in patients ≥ 90 years due to the rarity of this population in clinical trials. Our objective was to analyze the efficacy and toxicity of immune checkpoint inhibitors in patients ≥ 90 years. MATERIALS AND METHODS: We conducted a single center retrospective review of patients ≥ 90 years treated between July 2019 and September 2023 with standard of care ICIs for advanced urothelial cancer. RESULTS: Six patients treated with pembrolizumab were identified. Four (66.7%) were male and mean age was 93.5 years at the time of treatment initiation. Response rate was 66.7% (4 patients) with 3 complete responses, which were durable off therapy. Median follow up was 18.2 months. Median progression free survival (PFS) was 10.2 months [95%confidence interval (95%CI): 1.77, not reached (NR)] and median overall survival (OS) was 18.2 months (95%CI: 12.1, NR). Side effects presented in 4 (66.7%) patients and included hypothyroidism, diarrhea, anemia, thrombocytopenia, rash, and bullous dermatitis. One patient developed grade 3 anemia and no patients experienced grade 4 events or required hospitalization due to treatment side effects. CONCLUSIONS: Our experience in a small cohort of patients ≥ 90 years indicate that ICIs are well tolerated and effective for the treatment of advanced urothelial carcinoma in this patient population.

Comparison of clinicopathological characteristics, gene expression profiles, mutational analysis, and clinical outcomes of pure and mixed small-cell carcinoma of the bladder.

AIMS: Small cell bladder carcinoma (SCBC) is a rare, divergent form of urothelial carcinoma (UC). We aimed to determine whether pure (n = 16) and mixed (SCBC and UC; n = 30) tumours differed in pathology, gene expression characteristics, genetic alterations, and clinical outcomes. METHODS AND RESULTS: Forty (87%) patients received first-line chemotherapy. Twenty-nine patients had no metastatic disease at diagnosis and underwent radical cystectomy. There were no differences in age, sex, race distribution, tumour size, stage at presentation, therapy response with pathological downstaging to ≤ypT1N0, or overall or progression-free survival (PFS) between pure and mixed tumours. There was a longer PFS among downstaged chemotherapy-responding tumours ≤ypT2N0M0 than among unresponsive tumours ≥ypT2 ≥ yN1M1 (P = 0.001). Patients who achieved pathological downstaging with neoadjuvant chemotherapy (n = 10) were stage cT2N0M0 at the time of diagnosis and were alive at the last follow-up (median 37 months), while 46% of patients who failed to achieve pathological downstaging were alive at the last follow-up (median 38 months; P = 0.008). RNA sequencing showed that the UC of mixed SCBC had similar neural expression signatures to pure SCBC. DNA sequencing revealed alterations in TERT (83%), P53 (56%), ARID1A (28%), RB1 (22%), and BRCA2 (11%). Immunohistochemistry for RB1 showed loss of expression in 18/19 (95%) patients, suggesting frequent pathway downregulation despite a low prevalence of RB1 mutation. CONCLUSION: Patients with pure and mixed SCBC have similar outcomes and these outcomes are determined by the pathological stage at RC and are best among patients who have pathological downstaging after NAC.

BCG invokes superior STING-mediated innate immune response over radiotherapy in a carcinogen murine model of urothelial cancer.

Radiation and bacillus Calmette-Guérin (BCG) instillations are used clinically for treatment of urothelial carcinoma, but the precise mechanisms by which they activate an immune response remain elusive. The role of the cGAS-STING pathway has been implicated in both BCG and radiation-induced immune response; however, comparison of STING pathway molecules and the immune landscape following treatment in urothelial carcinoma has not been performed. We therefore comprehensively analyzed the local immune response in the bladder tumor microenvironment following radiotherapy and BCG instillations in a well-established spontaneous murine model of urothelial carcinoma to provide insight into activation of STING-mediated immune response. Mice were exposed to the oral carcinogen, BBN, for 12 weeks prior to treatment with a single 15 Gy dose of radiation or three intravesical instillations of BCG (1 × 108 CFU). At sacrifice, tumors were staged by a urologic pathologist and effects of therapy on the immune microenvironment were measured using the NanoString Myeloid Innate Immunity Panel and immunohistochemistry. Clinical relevance was established by measuring immune biomarker expression of cGAS and STING on a human tissue microarray consisting of BCG-treated non-muscle-invasive urothelial carcinomas. BCG instillations in the murine model elevated STING and downstream STING-induced interferon and pro-inflammatory molecules, intratumoral M1 macrophage and T-cell accumulation, and complete tumor eradication. In contrast, radiotherapy caused no changes in STING pathway or innate immune gene expression; rather, it induced M2 macrophage accumulation and elevated FoxP3 expression characteristic of immunosuppression. In human non-muscle-invasive bladder cancer, STING protein expression was elevated at baseline in patients who responded to BCG therapy and increased further after BCG therapy. Overall, these results show that STING pathway activation plays a key role in effective BCG-induced immune response and strongly indicate that the effects of BCG on the bladder cancer immune microenvironment are more beneficial than those induced by radiation. © 2021 The Pathological Society of Great Britain and Ireland.

Bone marrow-specific loss of ABI1 induces myeloproliferative neoplasm with features resembling human myelofibrosis.

Although the pathogenesis of primary myelofibrosis (PMF) and other myeloproliferative neoplasms (MPNs) is linked to constitutive activation of the JAK-STAT pathway, JAK inhibitors have neither curative nor MPN-stem cell-eradicating potential, indicating that other targetable mechanisms are contributing to the pathophysiology of MPNs. We previously demonstrated that Abelson interactor 1 (Abi-1), a negative regulator of Abelson kinase 1, functions as a tumor suppressor. Here we present data showing that bone marrow-specific deletion of Abi1 in a novel mouse model leads to development of an MPN-like phenotype resembling human PMF. Abi1 loss resulted in a significant increase in the activity of the Src family kinases (SFKs), STAT3, and NF-κB signaling. We also observed impairment of hematopoietic stem cell self-renewal and fitness, as evidenced in noncompetitive and competitive bone marrow transplant experiments. CD34+ hematopoietic progenitors and granulocytes from patients with PMF showed decreased levels of ABI1 transcript as well as increased activity of SFKs, STAT3, and NF-κB. In aggregate, our data link the loss of Abi-1 function to hyperactive SFKs/STAT3/NF-κB signaling and suggest that this signaling axis may represent a regulatory module involved in the molecular pathophysiology of PMF.

Intracranial cellular schwannomas: a clinicopathological study of 20 cases.

AIMS: Cellular schwannoma is a specific subtype of schwannoma, prone to misinterpretation as a malignant neoplasm. Involvement of the intracranial compartment by these tumours is extremely rare. We aim to characterise this clinicopathological subgroup. METHODS AND RESULTS: We identified a total of 20 cellular schwannomas with predominant intracranial involvement. The mean age of the patients at the time of surgery was 37 years (range = 16-81), with a slight female predominance (1.5:1 ratio). The most common sites were the eighth (n = 8) and fifth (n = 6) cranial nerves. Three tumours involved the anterior cranial fossa/olfactory groove, and a single case involved the glossopharyngeal nerve. All tumours met established criteria for cellular schwannoma, and were composed of interlacing fascicles of spindle cells lacking Verocay bodies with minimal Antoni B pattern and variable chronic inflammation and foamy histiocytes. Rare findings included haemosiderin deposition (n = 6), necrosis (n = 4), brisk mitotic activity (>10 mitoses per 10 high-power fields) (n = 2), focal epithelioid morphology (n = 2), myxoid areas (n = 2), neuroblastoma-like pattern (n = 1) and granular cells (n = 1). Immunohistochemical stains demonstrated expression of Schwann cell markers (S100 protein, SOX10, collagen IV) and preserved H3 K27 trimethylation in all cases tested. Fourteen patients had postoperative follow-up, ranging from 2 months to 21 years (mean = 66 months). In patients with follow-up, local recurrence/persistence developed in six cases; five tumours were initially incompletely resected. No metastatic disease or deaths were reported. CONCLUSIONS: Intracranial cellular schwannomas share morphological and immunophenotypical features with cellular schwannomas at others sites may demonstrate locally aggressive growth but appear to lack metastatic potential.

Prevalence of somatic and germline mutations of Fumarate hydratase in uterine leiomyomas from young patients.

AIMS: Hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome is caused by germline mutations in the Fumarate hydratase (FH) gene. In young women, the syndrome often presents with symptomatic uterine leiomyomas, leading to myomectomy or hysterectomy. In this study, we aimed to investigate the incidence and mutational profiles of FH-negative leiomyomas from young patients, thus allowing for early identification and triage of syndromic patients for surveillance. METHODS AND RESULTS: We evaluated 153 cases of uterine leiomyomas from women aged up to 30 years for loss of FH expression by tissue microarray (TMA)-based immunohistochemical staining. Mutational analysis of tumours with loss of FH was carried out by polymerase chain reaction (PCR) amplification of 10 exons within the FH gene and subsequent Sanger sequencing. The status of promoter methylation was assessed by bisulphite sequencing. Loss of FH protein expression was detected in seven (4.6%) of 153 tested uterine leiomyomas from young patients. All FH-negative leiomyomas displayed staghorn vasculature and fibrillary/neurophil-like cytoplasm. We found that six (86%) of seven FH-negative tumours detected by immunohistochemistry harboured FH mutations, 50% of which contained germline mutations. In particular, the germline mutational rate in FH gene was 2.0% (three of 153 cases). Bisulphite sequencing analysis failed to detect promoter methylation in any of the seven tumours. CONCLUSION: Our study showed a relatively high rate of FH germline mutation in FH-negative uterine leiomyomas from patients aged up to 30 years. While genetic mutations confer protein expression loss, epigenetic regulation of the FH gene appears to be unrelated to this phenotype.

Clinical significance of urothelial carcinoma ambiguous for muscularis propria invasion on initial transurethral resection of bladder tumor.

PURPOSE: To evaluate the clinical significance of invasive urothelial carcinoma that is ambiguous for muscularis propria invasion on initial transurethral resection of bladder tumor (TURBT). METHODS: All consecutive in-house TURBTs with invasive urothelial carcinoma from 1999 to 2017 that underwent radical cystectomy (RC) were grouped as follows: invasion of the lamina propria (INLP; n = 102; 24%), invasion of muscularis propria (INMP; n = 296; 69%) and ambiguous for muscularis propria invasion (AMP; n = 30; 7%). AMP was defined as extensive invasive carcinoma displaying thin muscle bundles where it is difficult to determine with certainty if those muscle bundles represent muscularis mucosae or muscularis propria (detrusor). Cases with any amount of small cell carcinoma or prior therapy were excluded. RESULTS: The average age was 66 years in INLP, 67 years in INMP, and 65 years in AMP. RC showed invasive carcinoma stage pT2 or above in 50/102 (49%) of INLP vs. 255/296 (86%) of INMP (P ≤ 001) vs. 25/30 (83.33%) of AMP (P = 0.002). Lymph nodes showed metastatic carcinoma in 18/98 (18.36%) of INLP vs. 96/272 (35.29%) of INMP (P = 0.002), and 6/25 (24%) in AMP (P = 0.729). The average follow-up was 48 months (range 0-192). Survival of AMP patients was similar to INLP and both were significantly better than INMP (P = 0.002 and P = 0.016). CONCLUSION: The great majority of patients with AMP on initial TURBT have advanced disease on RC and emphasizes the need for early repeat TURBT or even consideration of early cystectomy to lower the risk of worse pathological findings and to prolong survival.

A novel rat microsurgical model to study the immunological characteristics of male genital tissue in the context of penile transplantation.

Penis transplantation represents an exciting new avenue for restoration of male genitalia and function after devastating tissue loss. This animal model is designed to fill a critical void to study immunologic aspects related to reconstructive transplantation of male genitalia. A rat penile graft dissection was designed based on the internal pudendal arteries and dorsal penile vein and includes the skin of the prepuce. A nonsuture cuff technique was used to anastomose the graft vessels to the recipient superficial epigastric and femoral vessels. Seventy-seven penile transplantations were performed. Graft design yields suitable caliber and length of vessels at the radix of the penis. Anastomosis of the dorsal penile vein and the internal pudendal arteries insures optimal graft perfusion. The nonsuture cuff technique allows for successful microvascular anastomosis by a single surgeon with an average overall operative time of 2.5 h. Long-term graft survival (>30 days) was observed in syngeneic transplants. We have established a robust murine model with ideal vascular perfusion of penile tissue to study the unique immunobiology of male genitourinary allotransplantation. Heterotopic inset further allows for visual monitoring of graft viability, while the native penis serves as an optimal control.

Analysis of the Circulating Tumor Cell Capture Ability of a Slit Filter-Based Method in Comparison to a Selection-Free Method in Multiple Cancer Types.

Circulating tumor cells (CTCs) are a promising biomarker for cancer liquid biopsy. To evaluate the CTC capture bias and detection capability of the slit filter-based CTC isolation platform (CTC-FIND), we prospectively compared it head to head to a selection-free platform (AccuCyte®-CyteFinder® system). We used the two methods to determine the CTC counts, CTC positive rates, CTC size distributions, and CTC phenotypes in 36 patients with metastatic cancer. Between the two methods, the median CTC counts were not significantly different and the total counts were correlated (r = 0.63, p < 0.0001). The CTC positive rate by CTC-FIND was significantly higher than that by AccuCyte®-CyteFinder® system (91.7% vs. 66.7%, p < 0.05). The median diameter of CTCs collected by CTC-FIND was significantly larger (13.0 µm, range 5.2-52.0 vs. 10.4 µm, range 5.2-44.2, p < 0.0001). The distributions of CTC phenotypes (CK+EpCAM+, CK+EpCAM- or CK-EpCAM+) detected by both methods were similar. These results suggested that CTC-FIND can detect more CTC-positive cases but with a bias toward large size of CTCs.

Effects of a Thermal Accelerant Gel on Microwave Ablation Zone Volumes in Lung: A Porcine Study.

Background Thermal ablation of cancers may be associated with high rates of local tumor progression. A thermal accelerant gel has been developed to improve the transmission of microwave energy in biologic tissues with the aim of enlarging the thermal ablation zone. Purpose To determine the effects of a thermal accelerant gel on microwave ablation zone volumes in porcine lung and to compare percutaneous and endobronchial delivery methods. Materials and Methods Thirty-two consecutive microwave lung ablations were performed in nine 12-week-old domestic male swine under general anesthesia by using fluoroscopic guidance between September 2017 and April 2018. Experimental ablations were performed following percutaneous injection of thermal accelerant into the lung (n = 16) or after endobronchial injection by using a flexible bronchoscope (n = 8). Control ablations were performed without accelerant gel (n = 8). Lung tissue was explanted after the animals were killed, and ablation zone volumes were calculated as the primary outcome measure by using triphenyltetrazolium chloride vital staining. Differences in treatment volumes were analyzed by generalized mixed modeling. Results Thermal accelerant ablation zone volumes were larger than control ablations (accelerant vs control ablation, 4.3 cm3 [95% confidence interval: 3.4, 5.5] vs 2.1 cm3 [95% confidence interval: 1.4, 2.9], respectively; P < .001). Among ablations with the thermal accelerant, those performed following percutaneous injection had a larger average ablation zone volume than those performed following endobronchial injection (percutaneous vs endobronchial, 4.8 cm3 [95% confidence interval: 3.6, 6.4] vs 3.3 cm3 [95% confidence interval: 2.9, 3.8], respectively; P = .03). Ablation zones created after endobronchial gel injection were more uniform in size distribution (standard error, percutaneous vs endobronchial: 0.13 vs 0.07, respectively; P = .03). Conclusion Use of thermal accelerant results in larger microwave ablation zone volumes in normal porcine lung tissue. Percutaneous thermal accelerant injection leads to a larger ablation zone volume compared with endobronchial injection, whereas a more homogeneous and precise ablation zone size is observed by using the endobronchial approach. © RSNA, 2019 See also the editorial by Goldberg in this issue.

Role of immune microenvironment in gastrointestinal stromal tumours.

AIMS: The immune microenvironment is a prognostic factor for various malignancies. The significance of key players of this immune microenvironment, including tumour-infiltrating lymphocytes (TILs) and expression of programmed death-ligand 1 (PD-L1), indoleamine 2,3-dioxygenase (IDO) and tryptophanyl-tRNA synthetase (WARS) in gastrointestinal stromal tumours (GISTs) is largely unknown. METHODS AND RESULTS: Tissue microarrays were constructed from pathology files, 1996-2016. Immunohistochemistry for PD-L1, IDO and WARS was correlated with tumour size, mitoses and outcomes. TILs expressing CD3, CD4, CD8, FoxP3 and GBP5 were counted. A total of 129 GISTs were analysed. Mean patient age was 62.5 years; 52.0% were male. Tumour location included 89 stomach (69.0%), 33 small bowel (25.6%) and seven other (5.4%). Mean tumour size was 5.6 cm; mean mitoses were 7.2 per 50 high-power field. Nineteen patients (15.0%) developed disease progression, to abdominal wall (n = 8), liver (n = 6) and elsewhere (n = 5). Median progression-free survival was 56.6 months; five patients died of disease. PD-L1 was positive in 88 of 127 tumour samples (69.0%), 114 of 127 tumours were IDO-positive (89.8%) and 60 of 127 were positive for WARS (47.2%). PD-L1 was associated with increased size (P = 0.01), necrosis (P = 0.018) and mitoses (P = 0.006). Disease progression was not associated with PD-L1 (P = 0.44), IDO (P = 0.14) or WARS (P = 0.36) expression. PD-L1-positive GISTs with CD8+ or CD3+ TILs were significantly smaller than tumours with CD8+ or CD3+ TILs. CONCLUSIONS: PD-L1 expression was associated with increased size and mitoses. High CD8+ or CD3+ TIL counts were associated with decreased PD-L1/IDO+ GIST size. PD-L1 and IDO could be significant in GIST tumour biology, which invites consideration of immunotherapy as a potential treatment option.

Expression of PD-L1, indoleamine 2,3-dioxygenase and the immune microenvironment in gastric adenocarcinoma.

AIMS: The tumour microenvironment is increasingly important in several tumours. We studied the relationship of key players of immune microenvironment with clinicopathological parameters in gastric adenocarcinomas. METHODS AND RESULTS: Tissue microarrays were constructed from gastrectomy specimens, 2004-13. Immunohistochemistry was performed for programmed cell death ligand 1 (PD-L1), indoleamine 2,3-dioxygenase (IDO), tryptophanyl-tRNA synthetase (WARS), guanylate-binding protein 5 (GBP5), tumour-infiltrating lymphocytes (TIL) expressing CD3/CD8/FoxP3/PD1 and mismatch repair proteins (MMRs) MLH1, PMS2, MSH2 and MSH6. Clinicopathological parameters and clinical follow-up were recorded. The study included 86 patients; median follow-up was 34 months (0-148). Tumour types were 45% tubular, 38% diffuse, 17% mixed. PD-L1 was positive in 70%, epithelial IDO in 58%, stromal IDO in 91%, epithelial WARS in 67%, stromal WARS in 100%, epithelial GBP5 in 53% and stromal GBP5 in 71%. MMR-deficiency was found in 22%. There was no difference in biomarker expression by histological subtype, with the exception of fewer diffuse-type being MMR-deficient. Low stromal IDO was associated with decreased progression-free, overall and disease-specific survival. PD-L1-positive tumours were larger with MMR-deficiency and with increasing TILs, and had significantly higher FoxP3TILs. CONCLUSIONS: PD-L1 is expressed in a large proportion of gastric carcinomas, suggesting that therapy targeting this pathway could be relevant to many patients. PD-L1 expression and MMR-deficiency are associated with increased TILs and larger tumour size, emphasising their role in tumour biology. Higher stromal IDO expression is associated with better prognosis. Finally, we observed that immune modulators WARS and GBP5 are expressed highly in gastric adenocarcinomas, suggesting an important role in tumour pathobiology.

The in vivo performance of a novel thermal accelerant agent used for augmentation of microwave energy delivery within biologic tissues during image-guided thermal ablation: a porcine study.

OBJECTIVES: To investigate the effects of a novel caesium-based thermal accelerant (TA) agent on ablation zone volumes following in vivo microwave ablation of porcine liver and skeletal muscle, and to correlate the effects of TA with target organ perfusion. MATERIALS AND METHODS: This prospective study was performed following institutional animal care and use committee approval. Microwave ablation was performed in liver and resting skeletal muscle in eight Sus scrofa domesticus swine following administration of TA at concentrations of 0 mg/mL (control), 100 mg/mL and 250 mg/mL. Treated tissues were explanted and stained with triphenyltetrazolium chloride (TTC) for quantification of ablation zone volumes, which were compared between TA and control conditions. Hematoxylin and eosin (H&E) staining was also performed for histologic analysis. General mixed modelling with a log-normal distribution was used for all quantitative comparisons (p = 0.05). RESULTS: A total of 28 ablations were performed in the liver and 18 in the skeletal muscle. The use of TA significantly increased ablation zone volumes in a dose-dependent manner in both the porcine muscle and liver (p < 0.01). Both the absolute mean ablation zone volume and percentage increase in ablation zone volume were greater in the resting skeletal muscle than in the liver. In one swine, a qualitative mitigation of heat sink effects was observed by TTC and H&E staining. Non-lethal polymorphic ventricular tachycardia was identified in one swine, treated with intravenous amiodarone. CONCLUSIONS: The use of a novel TA agent significantly increased mean ablation zone volumes following microwave ablation using a porcine model. The relationship between TA administration and ablation size was dose-dependent and inversely proportional to the degree of target organ perfusion, and a qualitative reduction in heat-sink effects was observed.

Fatty acid-binding protein 1 is preferentially lost in microsatellite instable colorectal carcinomas and is immune modulated via the interferon γ pathway.

Fatty acid-binding protein 1 (FABP1) is an intracellular protein responsible for the transportation of long chain fatty acids. Aside from its functions in lipid metabolism and cellular differentiation, FABP1 also plays a role in inflammation through its interaction with peroxisome proliferator-activated receptors (PPARs). Previously, we compared expression of colonic epithelium genes in a subset of microsatellite instable (MSI) colorectal carcinomas (medullary carcinomas) to normal colonic mucosa and found that FABP1 expression was markedly decreased in the tumors. Further analysis of RNA expression in the colorectal subtypes and The Cancer Genome Atlas data set found that FABP1 expression is decreased in the CMS1 subset of colorectal carcinomas, which is characterized by microsatellite instability. As MSI colorectal carcinomas are known for their robust immune response, we then aimed to link FABP1 to the immune microenvironment of MSI carcinomas. To confirm the gene expression results, we performed immunohistochemical analysis of a cohort of colorectal carcinomas. FABP1 was preferentially lost in MSI carcinomas (123/133, 93%) compared with microsatellite stable carcinomas (240/562, 43%, P<0.0001). In addition, higher numbers of tumor-infiltrating lymphocytes were present in tumors with loss of FABP1 (P<0.0001). Decreased expression of the fatty acid storage and glucose regulator, PPARγ, was associated with the loss of FABP1 (P<0.0001). Colorectal cancer cell lines treated with interferon γ exhibited decreased expression of FABP1. FABP1 expression was partially recovered with the treatment of the cell lines with rosiglitazone, a PPARγ agonist. This study demonstrated that the loss of FABP1 expression is associated with MSI carcinomas and that interferon γ stimulation plays a role in this process via its interaction with PPARγ.

Differentiating tumor heterogeneity in formalin-fixed paraffin-embedded (FFPE) prostate adenocarcinoma tissues using principal component analysis of matrix-assisted laser desorption/ionization imaging mass spectral data.

RATIONALE: Many patients with adenocarcinoma of the prostate present with advanced and metastatic cancer at the time of diagnosis. There is an urgent need to detect biomarkers that will improve the diagnosis and prognosis of this disease. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is playing a key role in cancer research and it can be useful to unravel the molecular profile of prostate cancer biopsies. METHODS: MALDI imaging data sets are highly complex and their interpretation requires the use of multivariate statistical methods. In this study, MALDI-IMS technology, sequential principal component analysis (PCA) and two-dimensional (2-D) peak distribution tests were employed to investigate tumor heterogeneity in formalin-fixed paraffin-embedded (FFPE) prostate cancer biopsies. RESULTS: Multivariate statistics revealed a number of mass ion peaks obtained from different tumor regions that were distinguishable from the adjacent normal regions within a given specimen. These ion peaks have been used to generate ion images and visualize the difference between tumor and normal regions. Mass peaks at m/z 3370, 3441, 3447 and 3707 exhibited stronger ion signals in the tumor regions. CONCLUSIONS: This study reports statistically significant mass ion peaks unique to tumor regions in adenocarcinoma of the prostate and adds to the clinical utility of MALDI-IMS for analysis of FFPE tissue at a molecular level that supersedes all other standard histopathologic techniques for diagnostic purposes used in the current clinical practice. Copyright © 2016 John Wiley & Sons, Ltd.

Medullary carcinoma of the colon: a distinct morphology reveals a distinctive immunoregulatory microenvironment.

Medullary carcinoma of the colon is a unique histologic subtype of microsatellite unstable colorectal carcinoma but little is known regarding its tumor-immunoregulatory microenvironment. The aims of this study were to characterize the immune environment of medullary carcinoma and compare it with other microsatellite unstable and microsatellite stable colorectal carcinomas. An initial gene expression microarray analysis of six cases of medullary carcinoma was used to detect potentially differentially expressed genes. We extended this analysis utilizing genomic data from the Cancer Genome Atlas to compare eight cases of medullary carcinoma with other microsatellite unstable and stable carcinomas. Finally, we evaluated expression of key immune pathway proteins and lymphocyte subsets via immunohistochemistry of a large group of medullary carcinomas (n=105) and compared these findings with three other groups: poorly differentiated, microsatellite unstable well-differentiated and microsatellite stable well-differentiated carcinomas. Microarray and the Cancer Genome Atlas data analysis identified significant upregulation of several immunoregulatory genes induced by IFNγ including IDO-1, WARS (tRNA(trp)), GBP1, GBP4, GBP5, PDCD1 (PD-1), and CD274 (PD-L1) in medullary carcinoma compared with other microsatellite unstable and microsatellite stable tumors. By immunohistochemistry, IDO-1 was expressed in 64% of medullary carcinomas compared with 19% (9/47) of poorly differentiated carcinomas, 14% (3/22) of microsatellite unstable, and 7% (2/30) of the microsatellite stable well-differentiated carcinomas (P<0.0001). tRNA(trp) was overexpressed in 81% (84/104) of medullary carcinomas, 19% (9/47) of poorly differentiated, 32% (7/22) of microsatellite unstable, and 3% (1/30) of microsatellite stable well-differentiated carcinomas (P<0.0001). Medullary carcinoma had higher mean CD8+ and PD-L1+ tumor-infiltrating lymphocytes compared with all other groups (P<0.0001). This study demonstrates overexpression of several immunoregulatory genes in microsatellite unstable colorectal carcinomas and that expression of these genes and proteins is more prevalent in the medullary carcinoma subtype, which may be of use both diagnostically and therapeutically.

Tumour tissue-derived small extracellular vesicles reflect molecular subtypes of bladder cancer.

mRNA-based molecular subtypes have implications for bladder cancer prognosis and clinical benefit from certain therapies. Whether small extracellular vesicles (sEVs) can reflect bladder cancer molecular subtypes is unknown. We performed whole transcriptome RNA sequencing for formalin fixed paraffin embedded (FFPE) tumour tissues and sEVs separated from matched tissue explants, urine and plasma in patients with bladder cancer. sEVs were separated using size-exclusion chromatography, and characterized by transmission electron microscopy, nano flow cytometry and western blots, respectively. High yield of sEVs were obtained using approximately 1 g of tissue, incubated with media for 30 min. FFPE tumour tissue and tumour tissue-derived sEVs demonstrated good concordance in molecular subtype classification. All urinary sEVs were classified as luminal subtype, while all plasma sEVs were classified as Ba/Sq subtype, regardless of the molecular subtypes indicated by their matched FFPE tumour tissue. The comparison within urine sEVs, which may exclude the sample type specific background, could pick up the different biology between NMIBC and MIBC, as well as the signature genes related to molecular subtypes. Four candidate sEV-related bladder cancer-specific mRNA biomarkers, FAM71E2, OR4K5, FAM138F and KRTAP26-1, were identified by analysing matched urine sEVs, tumour tissue derived sEVs, and adjacent normal tissue derived sEVs. Compared to sEVs separated from biofluids, tissue-derived sEVs may reflect more tissue- or disease-specific biological features. Urine sEVs are promising biomarkers to be used for liquid biopsy-based molecular subtype classification, but the current algorithm needs to be modified/adjusted. Future work is needed to validate the four new bladder cancer-specific biomarkers in large cohorts.

Development and Validation of a Multivariable Nomogram Predictive of Post-Nephroureterectomy Renal Function.

BACKGROUND AND OBJECTIVE: The timing of perioperative nephrotoxic chemotherapy for upper tract urothelial carcinoma (UTUC) remains controversial and strongly depends on predicted platinum eligibility after radical nephroureterectomy (RNU). The study objective was to develop and validate a multivariable nomogram to predict estimated glomerular filtration rate (eGFR) following RNU. METHODS: This was a multi-institutional retrospective study of patients with UTUC treated with RNU from 2000 to 2020 at seven high-volume referral centers. Use of adjuvant chemotherapy was risk-stratified. Patients were retrospectively randomly allocated 2:1 to discovery and validation cohorts. Discovery data were used to identify independent factors associated with GFR at 1-3 mo after RNU on linear regression, and backward selection was applied for model construction. Accuracy was defined as the percentage of predicted eGFR results within 30% of the corresponding observed eGFR. KEY FINDINGS AND LIMITATIONS: We included 1100 patients, of whom 733 were in the discovery and 367 were in the validation cohort. Multivariable predictors of postoperative eGFR decline included advanced age (odds ratio [OR] -0.18, 95% confidence interval [CI] -0.28 to -0.08), diabetes (OR -2.38, 95% CI -4.64 to -0.11), and hypertension (OR -2.24, 95% CI -4.16 to -0.32). Factors associated with favorable postoperative eGFR included larger tumor size (OR 10.57, 95% CI 7.4-13.74 for tumors >5 cm vs ≤2 cm) and preoperative eGFR (OR 0.44, 95% CI 0.39-0.49). A composite nomogram predicted postoperative eGFR with good accuracy in both the discovery (80.5%) and validation (78.6%) cohorts. Limitations include exclusion of patients who received neoadjuvant chemotherapy. CONCLUSIONS: A nomogram that incorporates ubiquitous preoperative clinical variables can predict post-RNU eGFR and was validated with an independent cohort. PATIENT SUMMARY: We developed a tool that uses patient data to predict eligibility for chemotherapy after surgery to remove the kidney and ureter in patients with cancer in the upper urinary tract.

Metabolic Reprogramming of Tumor-Associated Macrophages Using Glutamine Antagonist JHU083 Drives Tumor Immunity in Myeloid-Rich Prostate and Bladder Cancers.

Glutamine metabolism in tumor microenvironments critically regulates antitumor immunity. Using the glutamine-antagonist prodrug JHU083, we report potent tumor growth inhibition in urologic tumors by JHU083-reprogrammed tumor-associated macrophages (TAMs) and tumor-infiltrating monocytes. We show JHU083-mediated glutamine antagonism in tumor microenvironments induced by TNF, proinflammatory, and mTORC1 signaling in intratumoral TAM clusters. JHU083-reprogrammed TAMs also exhibited increased tumor cell phagocytosis and diminished proangiogenic capacities. In vivo inhibition of TAM glutamine consumption resulted in increased glycolysis, a broken tricarboxylic acid (TCA) cycle, and purine metabolism disruption. Although the antitumor effect of glutamine antagonism on tumor-infiltrating T cells was moderate, JHU083 promoted a stem cell-like phenotype in CD8+ T cells and decreased the abundance of regulatory T cells. Finally, JHU083 caused a global shutdown in glutamine-utilizing metabolic pathways in tumor cells, leading to reduced HIF-1α, c-MYC phosphorylation, and induction of tumor cell apoptosis, all key antitumor features. Altogether, our findings demonstrate that targeting glutamine with JHU083 led to suppressed tumor growth as well as reprogramming of immunosuppressive TAMs within prostate and bladder tumors that promoted antitumor immune responses. JHU083 can offer an effective therapeutic benefit for tumor types that are enriched in immunosuppressive TAMs.

Immunomodulatory response to neoadjuvant nivolumab in non-metastatic clear cell renal cell carcinoma.

Novel perioperative strategies are needed to reduce recurrence rates in patients undergoing nephrectomy for high-risk, non-metastatic clear cell renal cell carcinoma (ccRCC). We conducted a prospective, phase I trial of neoadjuvant nivolumab prior to nephrectomy in 15 evaluable patients with non-metastatic ccRCC. We leveraged tissue from that cohort to elucidate the effects of PD-1 inhibition on immune cell populations in ccRCC and correlate the evolving immune milieu with anti-PD-1 response. We found that nivolumab durably induces a pro-inflammatory state within the primary tumor, and baseline immune infiltration within the primary tumor correlates with nivolumab responsiveness. Nivolumab increases CTLA-4 expression in the primary tumor, and subsequent nephrectomy increases circulating concentrations of sPD-L1, sPD-L3 (sB7-H3), and s4-1BB. These findings form the basis to consider neoadjuvant immune checkpoint inhibition (ICI) for high-risk ccRCC while the tumor remains in situ and provide the rationale for perioperative strategies of novel ICI combinations.