Expression and Prognostic Significance of LAG-3, TIGIT, VISTA, and IDO1 in Endometrial Serous Carcinoma.

Endometrial serous carcinoma (ESC) is an uncommon, aggressive type of endometrial cancer. While immune checkpoint blockade has emerged as a promising treatment option for endometrial carcinomas, research on the expression of immune checkpoints that could serve as prospective immunotherapy targets in ESC is limited. We examined the prevalence and prognostic value of lymphocyte-activation gene 3 (LAG-3), T-cell immunoglobulin and ITIM domain (TIGIT), V-domain immunoglobulin (Ig) suppressor of T-cell activation (VISTA), and indoleamine 2,3-dioxygenase 1 in 94 cases of ESC and correlated their expression with CD8+ and FOXP3+ tumor-infiltrating lymphocytes (TILs). We observed a positive correlation among LAG-3, TIGIT, and VISTA expressed on immune cells, and among these markers and CD8+ and FOXP3+ TIL densities. In Kaplan-Meier survival analysis, tumors with high levels of LAG-3 and TIGIT expression had better progression-free survival (PFS) and overall survival (OS) than those with lower levels of expression (LAG-3: PFS, P = .03, OS, P = .04; TIGIT: PFS, P = .01, OS, P = .009). In multivariate analysis, only high TIGIT expression was of independent prognostic value for better OS. VISTA expression in immune or tumor cells, and indoleamine 2,3-dioxygenase 1 expression in tumor cells, did not show a significant association with survival. Our data indicate that LAG-3, TIGIT, and VISTA immune checkpoints have roles in the microenvironment of ESC, and their expression patterns highlight the complex interactions among the different components of this system. High levels of these markers, together with high CD8+ TIL, suggest the potential immunogenicity of a subset of these tumors. Further studies are needed to elucidate the roles of various immune components in the ESC microenvironment and their association with intrinsic tumor properties.

Histopathology Based AI Model Predicts Anti-Angiogenic Therapy Response in Renal Cancer Clinical Trial.

Background: Predictive biomarkers of treatment response are lacking for metastatic clearcell renal cell carcinoma (ccRCC), a tumor type that is treated with angiogenesis inhibitors, immune checkpoint inhibitors, mTOR inhibitors and a HIF2 inhibitor. The Angioscore, an RNA-based quantification of angiogenesis, is arguably the best candidate to predict anti-angiogenic (AA) response. However, the clinical adoption of transcriptomic assays faces several challenges including standardization, time delay, and high cost. Further, ccRCC tumors are highly heterogenous, and sampling multiple areas for sequencing is impractical. Approach: Here we present a novel deep learning (DL) approach to predict the Angioscore from ubiquitous histopathology slides. In order to overcome the lack of interpretability, one of the biggest limitations of typical DL models, our model produces a visual vascular network which is the basis of the model's prediction. To test its reliability, we applied this model to multiple cohorts including a clinical trial dataset. Results: Our model accurately predicts the RNA-based Angioscore on multiple independent cohorts (spearman correlations of 0.77 and 0.73). Further, the predictions help unravel meaningful biology such as association of angiogenesis with grade, stage, and driver mutation status. Finally, we find our model is able to predict response to AA therapy, in both a real-world cohort and the IMmotion150 clinical trial. The predictive power of our model vastly exceeds that of CD31, a marker of vasculature, and nearly rivals the performance (c-index 0.66 vs 0.67) of the ground truth RNA-based Angioscore at a fraction of the cost. Conclusion: By providing a robust yet interpretable prediction of the Angioscore from histopathology slides alone, our approach offers insights into angiogenesis biology and AA treatment response.

Cost analyses for malaria molecular diagnosis for research planners in India and beyond.

BACKGROUND: Malaria elimination mandates early and accurate diagnosis of infection. Although malaria diagnosis is programmatically dependent on microscopy/RDTs, molecular diagnosis has much better diagnostic accuracy. Higher cost of molecular diagnoses is a recognized challenge for use at the point of care. Because funding is always a recognized constraint, we performed financial cost-analyses of available molecular platforms for better utilization of available budget. METHODS: Two strategies were applied to deduce the cost per sample. Strategy 1 included recurring components (RC) in minimum pack size, and biologist's time whereas strategy 2 included only RC and non-recurring components and costs are calculated for sample sizes (1-1,000,000) to infer the sample size effect. RESULTS: Spin column-based manual DNA extraction (US$ 3.93 per sample) is the lowest-cost method, followed by magnetic bead-based automated, semi-automated, and PCI-based manual method. Further, DNA extraction cost per sample via spin column-based manual method and semi-automated method decreases with an increase in sample size up to 10,000. Real-time PCRs are ~ 2-fold more economical than conventional PCR, regardless of sample size. CONCLUSIONS: This study is the first for malaria to estimate systematic molecular diagnosis financial costs. Kit-based and automated methods may replace conventional DNA extraction and amplification methods for a frugal high-throughput diagnosis.

COL1A1-PDGFB Fusion Associated Fibrosarcoma of the Uterine Corpus: A Case Report and Literature Review.

Uterine collagen type 1 alpha 1 (COL1A1) and platelet-derived growth factor beta chain (PDGFB) fusion associated fibrosarcoma is a recently described entity characterized by a specific translocation t(17;22) (q22;q13) leading to the formation of COL1A1-PDGFB fusion transcripts that are typically associated with dermatofibrosarcoma protuberans. So far, only 4 cases of COL1A1-PDGFB fusion associated fibrosarcoma involving the female reproductive system have been reported in the literature. All cases showed strong diffuse expression of CD34. COL1A1-PDGFB fusion associated fibrosarcomas are aggressive tumors with a propensity for chemotherapy resistance and a poor prognosis. We are reporting the fifth case of a uterine COL1A1-PDGFB fusion associated fibrosarcoma. A 58-yr-old female presented with a large uterine mass with extension into bilateral pelvic sidewalls, mesentery of rectosigmoid colon and the vagina. A hysterectomy, bilateral salpingo-oophorectomy, and tumor debulking procedure was performed. Microscopic evaluation revealed a mitotically active cellular spindle cell neoplasm with focal osteoclast like giant cells, myxoid changes and necrosis. CD34 was diffusely and strongly positive throughout the tumor. Next-generation sequencing showed presence of the COL1A1-PDGFB fusion. The patient was treated with multiple chemotherapy regimens, however, progressed under therapy with worsening symptoms and development of extensive pelvic disease. She died of disease 13 mo after the initial diagnosis. In summary, uterine COL1A1-PDGFB fusion associated fibrosarcomas are rare tumors with aggressive clinical behavior that need to be considered in the differential diagnosis of CD34-positive uterine spindle cell neoplasms. Novel treatment options may include imatinib, a tyrosine kinase inhibitor used for treatment of advanced and unresectable dermatofibrosarcoma protuberans that was given in 1 uterine sarcoma case and showed promising initial response.

Intratumoral Resolution of Driver Gene Mutation Heterogeneity in Renal Cancer Using Deep Learning.

Intratumoral heterogeneity arising from tumor evolution poses significant challenges biologically and clinically. Dissecting this complexity may benefit from deep learning (DL) algorithms, which can infer molecular features from ubiquitous hematoxylin and eosin (H&E)-stained tissue sections. Although DL algorithms have been developed to predict some driver mutations from H&E images, the ability of these DL algorithms to resolve intratumoral mutation heterogeneity at subclonal spatial resolution is unexplored. Here, we apply DL to a paradigm of intratumoral heterogeneity, clear cell renal cell carcinoma (ccRCC), the most common type of kidney cancer. Matched IHC and H&E images were leveraged to develop DL models for predicting intratumoral genetic heterogeneity of the three most frequently mutated ccRCC genes, BAP1, PBRM1, and SETD2. DL models were generated on a large cohort (N = 1,282) and tested on several independent cohorts, including a TCGA cohort (N = 363 patients) and two tissue microarray (TMA) cohorts (N = 118 and 365 patients). These models were also expanded to a patient-derived xenograft (PDX) TMA, affording analysis of homotopic and heterotopic interactions of tumor and stroma. The status of all three genes could be inferred by DL, with BAP1 showing the highest sensitivity and performance within and across tissue samples (AUC = 0.87-0.89 on holdout). BAP1 results were validated on independent human (AUC = 0.77-0.84) and PDX (AUC = 0.80) cohorts. Finally, BAP1 predictions correlated with clinical outputs such as disease-specific survival. Overall, these data show that DL models can resolve intratumoral heterogeneity in cancer with potential diagnostic, prognostic, and biological implications. SIGNIFICANCE: This work demonstrates the potential for deep learning analysis of histopathologic images to serve as a fast, low-cost method to assess genetic intratumoral heterogeneity. See related commentary by Song et al., p. 2672.

MYC Amplification in Epithelioid Angiosarcoma of the Urinary Bladder and Prostate Following Prostate Radiotherapy: A Case Report with a Novel Molecular Alteration.

Epithelioid angiosarcoma is a rare variant of angiosarcoma. Radiation-associated epithelioid angiosarcoma of the urinary bladder and prostate is an exceedingly rare tumor and there are only 8 cases of epithelioid angiosarcoma of the urinary bladder and prostate associated with previous radiotherapy in the literature. To the best of our knowledge, MYC gene amplification has not been previously reported in epithelioid angiosarcoma of the urinary bladder and prostate following radiotherapy, although it is observed in radiation-associated angiosarcoma of other anatomic sites. Here we report the first case of epithelioid angiosarcoma of the urinary bladder and prostate with MYC gene amplification detected by fluorescence in situ hybridization (FISH) analysis in a 70-year-old male patient 10 years after receiving radiation and hormonal therapy for prostate cancer.

Retroperitoneal Sclerosing Angiomyolipoma with Long-Term Follow up: A Case Report with Unique Clinicopathologic and Genomic Profile.

Sclerosing angiomyolipoma (sAML) is a rare variant of the perivascular epithelioid tumors exhibiting distinct morphology with extensive stromal hyalinization, which makes it challenging to recognize. It often lacks an adipose tissue component and melanocytic markers may be expressed only focally, further posing a diagnostic challenge. Here, we report a case of sAML of the left pararenal retroperitoneum in a 52-year-old woman with 92 months of clinical follow up and discuss the histologic features, immunoprofile, molecular alterations, and differential diagnoses that can aid in the diagnosis of this unique and rare entity.

SPARC is a key mediator of TGF-ß-induced renal cancer metastasis.

Aberrant expression of transforming growth factor-ß1 (TGF-ß1) is associated with renal cell carcinoma (RCC) progression by inducing cancer metastasis. However, the downstream effector(s) in TGF-ß signaling pathway is not fully characterized. In the present study, the elevation of secreted protein acidic and rich in cysteine (SPARC) as a TGF-ß regulated gene in RCC was identified by applying differentially expressed gene analysis and microarray analysis, we further confirmed this result in several RCC cell lines. Clinically, the expression of these two genes is positively correlated in RCC patient specimens. Furthermore, elevated SPARC expression is found in all the subtypes of RCC and positively correlated with the RCC stage and grade. In contrast, SPARC expression is inversely correlated with overall and disease-free survival of patients with RCC, suggesting SPARC as a potent prognostic marker of RCC patient survival. Knocking down SPARC significantly inhibits RCC cell invasion and metastasis both in vitro and in vivo. Similarly, in vitro cell invasion can be diminished by using a specific monoclonal antibody. Mechanistically, SPARC activates protein kinase B (AKT) pathway leading to elevated expression of matrix metalloproteinase-2 that can facilitate RCC invasion. Altogether, our data support that SPARC is a critical role of TGF-ß signaling network underlying RCC progression and a potential therapeutic target as well as a prognostic marker.

PTRF independently predicts progression and survival in multiracial upper tract urothelial carcinoma following radical nephroureterectomy.

OBJECTIVES: Polymerase I and transcript release factor (PTRF) has been implicated in cancer biology but its role in upper tract urothelial carcinoma (UTUC) is unknown. From a pilot transcriptome, we identified PTRF was significantly upregulated in high stage UTUC. Bladder cancer transcriptome from The Cancer Genome Atlas (TCGA) supported our finding and high PTRF level also predicted poor survival. We, therefore, investigated the correlation of PTRF with patients' clinicopathologic characteristics and outcomes in a multiracial UTUC cohort. MATERIALS AND METHODS: By immunohistochemical staining, PTRF expression was determined using H-score. PTRF expression of 575 UTUCs from 8 institutions, including 118 Asians and 457 Caucasians, was compared with various clinicopathologic parameters. Human urothelial cancer cell lines were used to evaluate the level of PTRF protein and mRNA expression, and PTRF transcript level was assessed in fresh samples from 12 cases of the cohort. The impact of PTRF expression on disease progression, cancer-specific death and overall mortality was also examined. RESULTS: High PTRF expression was significantly associated with multifocality (P = 0.023), high pathologic tumor stage (P < 0.00001), nonurothelial differentiation (P = 0.035), lymphovascular invasion (P = 0.003) and lymph node metastasis (P = 0.031). PTRF mRNA expression was also markedly increased in advanced stage UTUC (P = 0.0003). High PTRF expressing patients had consistently worse outcomes than patients with low PTRF expression regardless of demographic variation (all P < 0.005). In multivariate analysis, high PTRF expression was an independent predictor for progression-free survival (hazard ratio [HR] 1.70, 95% confidence interval [CI] 1.07-2.69, P = 0.025), cancer-specific survival (HR 2.09, 95% CI 1.28-3.42, P = 0.003), and overall survival (HR 2.04, 95% CI 1.33-3.14, P = 0.001). CONCLUSIONS: Results indicate that PTRF is a predictive biomarker for progression and survival and an independent prognosticator of UTUC. Elevated PTRF could probably propel clinically aggressive disease and serve as a potential therapeutic target for UTUC.

Expression and prognostic utility of PD-L1 in patients with squamous cell carcinoma of the bladder.

OBJECTIVES: Checkpoint inhibitors are approved for the treatment of urothelial bladder cancer. However, there have been no reports on the prognostic value of programmed-death receptor ligand 1 (PD-L1) expression in squamous cell carcinoma (SCC) of the bladder. We assessed the relationship between PD-L1 expression, clinicopathological features, and oncologic outcomes in bladder SCC. METHODS AND MATERIALS: Immunohistochemistry of PD-L1 was performed on 151 radical cystectomy specimens with pure SCC treated in Mansoura, Egypt from 1997 to 2004. RESULTS: Median patient age was 52 years (range: 36-74 years) and median length of follow up was 63 months (range: 1-100 months). Schistosomiasis was present in 81% of the specimens and 93% had muscle-invasive disease on pathologic staging. PD-L1 expression was negative in 50 (33%) of the specimens. Negative PD-L1 expression was associated with higher pathologic tumor stage (P = 0.04), higher grade lesions (P = 0.01), and the presence of lymphovascular invasion (P < 0.01). Kaplan-Meier analyses showed that negative PD-L1 expression is associated with worse recurrence-free (P = 0.01) and worse cancer-specific survival (P = 0.01). Multivariable Cox regression analyses showed negative PD-L1 expression was an independent predictor of disease recurrence (hazards ratio 2.05, 95% confidence interval 1.06-3.96, P = 0.03) and cancer-specific mortality (hazards ratio 2.89, 95% confidence interval 1.22-6.82, P = 0.02). CONCLUSIONS: Negative PD-L1 expression is associated with higher pathologic tumor stage, higher grade lesions, presence of lymphovascular invasion, and worse oncologic outcomes after radical cystectomy for SCC. These findings support the need for the inclusion of patients with bladder SCC into immunotherapy clinical trials.

Prognostic significance of BAP1 expression in high-grade upper tract urothelial carcinoma: a multi-institutional study.

PURPOSE: To evaluate the prognostic value of BRCA1-associated protein-1 (BAP1) expression in upper tract urothelial carcinoma (UTUC), as BAP1 mutations have been associated with prognostic implications in urologic and non-urologic malignancies. METHODS: We reviewed a multi-institutional cohort of patients who underwent radical nephroureterectomy (RNU) for high-grade UTUC from 1990-2008. Immunohistochemistry (IHC) for BAP1 was performed on tissue microarrays. Staining intensity was graded from 0-3, with BAP1 loss defined as an average intensity of < 1. Clinicopathologic characteristics and oncologic outcomes [recurrencefree (RFS), cancer-specific (CSS), and overall survival (OS)] were stratified by BAP1 status. The prognostic role of BAP1 was assessed using Kaplan-Meier (KM) and Cox regression analysis. Significance was defined as p < 0.05. RESULTS: 348 patients were included for analysis and 173 (49.7%) showed BAP1 loss. Median follow-up was 36.0 months. BAP1 loss was associated with papillary architecture and absence of tumor necrosis or CIS. On univariable analysis, BAP1 loss was associated with improved RFS (HR 0.60, p = 0.013) and CSS (HR 0.55, p = 0.007), although significance was lost on multivariable analysis (HR 0.71, p = 0.115 and HR 0.65, p = 0.071; respectively) after adjusting for other significant parameters. BAP1 expression was not significantly associated with OS. CONCLUSIONS: BAP1 loss was associated with favorable pathologic features and better oncologic outcomes in univariate but not multivariate analysis in patients with high-grade UTUC. In contrast to renal cell carcinoma, loss of BAP1 expression appears to confer a better prognosis in high-grade UTUC. The role of the BAP1 pathway in UTUC pathogenesis remains to be further elucidated.

Multi-institutional evaluation of the prognostic significance of EZH2 expression in high-grade upper tract urothelial carcinoma.

PURPOSE: Enhancer of zeste homolog 2 is a methyltransferase encoded by the EZH2 gene, whose role in upper tract urothelial carcinoma (UTUC) is poorly understood. We sought to evaluate the prognostic value of EZH2 expression in UTUC. METHODS: We reviewed a multi-institutional cohort of patients who underwent radical nephroureterectomy for high-grade UTUC from 1990 to 2008. Immunohistochemistry for EZH2 was performed on tissue microarrays. Percentage of staining was evaluated, and the discriminative value of EZH2 was tested, with EZH2 positivity defined as>20% staining present. Clinicopathologic characteristics and oncologic outcomes (recurrence-free (RFS), cancer-specific (CSS), and overall survival (OS)) were compared, stratified by EZH2 positivity. The prognostic role of EZH2 was assessed using Kaplan-Meier, univariate (UVA), and multivariate (MVA) Cox regression analyses. Significance was defined for P<0.05. RESULTS: A total of 376 patients were included for analysis, with median follow-up 36.0 months. Overall, 78 (20.7%) were EZH2-positive. EZH2 expression was more often associated with ureteral location, lymphovascular invasion, sessile architecture, necrosis, and concomitant carcinoma in situ. On UVA, increased EZH2 expression was a significant predictor for inferior RFS (HR 1.63, P = 0.033), CSS (HR 2.03, P = 0.003), and OS (HR 2.11, P<0.001). On MVA EZH2 remained a significant predictor of worse CSS (HR 1.99 [95% CI: 1.21-3.27], P = 0.007) and OS (HR 1.54 [95% CI: 1.06-2.24], P = 0.024), while significance was lost for RFS. CONCLUSION: Increased EZH2 expression is associated with adverse pathologic features and inferior oncologic outcomes in patients with high-grade UTUC. The role of EZH2 biology in UTUC pathogenesis remains to be further elucidated.

Multicenter Validation of Enhancer of Zeste Homolog 2 Expression as an Independent Prognostic Marker in Localized Clear Cell Renal Cell Carcinoma.

Purpose Enhancer of zeste homolog 2 (EZH2), a chromatin remodeler, is implicated in the pathogenesis of clear cell renal cell carcinoma (ccRCC). However, the effect of EZH2 on outcomes in localized ccRCC is unclear, and molecular biomarkers are not currently integrated into prognostic models or adjuvant therapy trials. Methods We performed Cox regression to evaluate the association of tumor-based EZH2 gene and protein expression with survival in three independent cohorts: a cohort from The Cancer Genome Atlas (n = 532), a cohort from University of Texas Southwestern Medical Center (n = 122), and a cohort from Mayo Clinic (n = 1,338). Analyses were adjusted for the prognostic stage, size, grade, and necrosis (SSIGN) score as well as within low-, intermediate-, and high-risk SSIGN groups. Results Patients in The Cancer Genome Atlas cohort with EZH2-high gene expression were 1.5 times more likely to experience overall death than patients with EZH2-low expression (95% CI, 1.1 to 2.3; P = .028). Patients in the University of Texas Southwestern Medical Center cohort with EZH2-high protein expression were two times more likely to experience overall death than patients with EZH2-low expression (95% CI, 1.1 to 4.4; P = .034). Similarly, patients in the Mayo Clinic cohort with EZH2-high protein expression were 1.4 times more likely to experience overall death (95% CI, 1.2 to 1.7; P < .001). Patients in the Mayo Clinic cohort with EZH2-high protein expression were nearly two times more likely to experience RCC-specific death (95% CI, 1.5 to 2.6; P < .001); EZH2 protein expression was particularly prognostic among patients with low-risk SSIGN tumors (HR, 6.1; 95% CI, 3.4 to 11.1; P < .001). Conclusion EZH2 expression accurately predicts risk of RCC death beyond existing clinicopathologic models, particularly in low- and intermediate-risk SSIGN tumors. Further studies are required to incorporate molecular biomarkers into surveillance guidelines and adjuvant clinical trials.

Probiotics: Potent Immunomodulatory Tool Against Allergy.

Probiotics are defined as live microbial food ingredients that produce several beneficial effects to human health. Probiotic bacteria have been mostly investigated in the prevention of and treatment for different gastrointestinal diseases and allergies. It is not fully clear how probiotics exert their beneficial effects on health, but one of the most probable mechanisms of action is the modulation of immune responses via the mucosal immune system of the gut. Commensal bacteria in the gastrointestinal tract play an integral role in both innate and humoral immunity. It is well established that this protective role can be maintained or modulated by the ingestion of probiotics. More recently, it has been shown that specific probiotic strains can influence the secretion of cytokines to help direct naïve T-helper cells toward either a Th1-dominant, cell-mediated immune response or toward a Th2-dominant, humoral immune response. This paper will review current knowledge of the Th1/Th2 model of humoral immunity as well as introduce how strain-specific probiotics can be used therapeutically to help balance this immune response and therefore help prevent allergy.