Quantitative Phosphoproteomics Analysis Uncovers PAK2- and CDK1-Mediated Malignant Signaling Pathways in Clear Cell Renal Cell Carcinoma.

Clear cell Renal Cell Carcinoma (ccRCC) is among the 10 most common cancers in both men and women and causes more than 140,000 deaths worldwide every year. In order to elucidate the underlying molecular mechanisms orchestrated by phosphorylation modifications, we performed a comprehensive quantitative phosphoproteomics characterization of ccRCC tumor and normal adjacent tissues. Here, we identified 16,253 phosphopeptides, of which more than 9000 were singly quantified. Our in-depth analysis revealed 600 phosphopeptides to be significantly differentially regulated between tumor and normal tissues. Moreover, our data revealed that significantly up-regulated phosphoproteins are associated with protein synthesis and cytoskeletal re-organization which suggests proliferative and migratory behavior of renal tumors. This is supported by a mesenchymal profile of ccRCC phosphorylation events. Our rigorous characterization of the renal phosphoproteome also suggests that both epidermal growth factor receptor and vascular endothelial growth factor receptor are important mediators of phospho signaling in RCC pathogenesis. Furthermore, we determined the kinases p21-activated kinase 2, cyclin-dependent kinase 1 and c-Jun N-terminal kinase 1 to be master kinases that are responsible for phosphorylation of many substrates associated with cell proliferation, inflammation and migration. Moreover, high expression of p21-activated kinase 2 is associated with worse survival outcome of ccRCC patients. These master kinases are targetable by inhibitory drugs such as fostamatinib, minocycline, tamoxifen and bosutinib which can serve as novel therapeutic agents for ccRCC treatment.

The clinical predictive factors and postoperative histopathological parameters associated with upgrading after radical prostatectomy: A contemporary analysis with grade groups.

BACKGROUND AND AIM: Upgrading after radical prostatectomy (RP) is an ongoing problem since first description of Gleason score. In this retrospective study, our aim is to investigate upgrading after RP in grade groups (GG) and clinical predictive, and postoperative histopathological factors associated with GG upgrading (GGU). PATIENTS AND METHODS: A total of 753 patients undergoing RP between January 2006 and June 2019 at our institution were investigated. Overall cohort were divided into two groups according to GGU status after RP as nonupgrading and upgrading. Retrospectively documented preoperative clinical and postoperative histopathological parameters were compared between two groups. Furthermore, we investigated a subgroup of institutional cohort (n = 398) whose prostate biopsy (Pbx) and RP were performed in our institution and we also divided this cohort into two groups according to GGU status. χ2 and Mann-Whitney U tests were used for comparative analyses. The independent preoperative predictive and postoperative histopathological factors associated with GGU were investigated using multivariate logistic regression analysis. RESULTS: The total GGU was 55.8% in overall cohort and 45.2% in institutional cohort. The GGU was found as the most common in bioptic GG1 group in both overall (64.0%), and institutional (54.5%) cohorts. In multivariate analyses, the noninstitutional Pbx (odds ratio [OR] = 2.56; 95% confidence interval [CI]: 1.86-3.51; P < .001), tumor positive core numbers in Pbx (OR = 1.11; 95%CI: 1.04-1.19; P = .003), increased prostate specific antigen (PSA) density (OR = 3.59; 95%CI: 1.03-12.52, P = .045) and age (OR = 1.03; 95%CI: 1.00-1.05, P = .046) were independent clinical predictors of GGU in overall cohort whereas only increased PSA density (OR = 5.94; 95%CI: 1.28-27.50; P = .023) was independent predictor in institutional cohort. Among postoperative histopathological factors, perineural invasion (OR = 1.57; 95%CI: 1.70-3.87; P < .001 and OR = 2.53; 95%CI: 1.46-4.40; P = .001, respectively), increased maximum tumor diameter (OR = 1.46; 95%CI: 1.23-1.73; P < .001 and OR = 1.33; 95%CI: 1.07-1.66; P = .010, respectively), and high-grade prostatic intraepithelial neoplasia (HGPIN) existence at tumor surrounding tissue (OR = 1.96; 95%CI: 1.32-2.90; P = .001 and OR = 1.87; 95%CI: 1.10-3.21; P = .022, respectively) were independently associated with GGU after RP, in both of overall and institutional cohorts. CONCLUSIONS: Noninstitutional prostate biopsy, increased PSA density, higher tumor positive cores in Pbx and older age are the clinical predictors of upgrading after RP in contemporary GG. Perineural invasion, increased maximum tumor diameter, and HGPIN existence at tumor surrounding tissue are postoperative histopathological factors associated with GGU.

IDO1-mediated catabolism of tryptophan in gastric tumors: Its potential role in the axis of histopathology, differentiation and metastasis.

BACKGROUND: Indoleamine 2,3-Dioxygenase 1 (IDO1)-mediated tryptophan degradation, which is the rate-limiting enzyme of tryptophan/kynurenine pathway, may cause immune suppression in the tumor microenvironment, while potentiating proliferative and metastatic activity in the tumor focus, Phase studies of IDO1 inhibitors are ongoing, and our study aims to evaluate the potential contribution of IDO1 gene expression to the tryptophan/kynurenine pathway in tumor and tumor microenvironment foci in gastric cancer (GC) on a clinicopathological axis, METHOD: In the case-control study design, the determination of tryptophan and its metabolites in the serum of 51 GC and 49 healthy controls was made using High Pressure Liquid Chromatography-Fluorescence Detector (HPLC-FD). IDO1 expression in a total of 102 tissues with tumor and tumor microenvironment was detected by quantitative PCR (q-PCR). RESULTS: In gastric tumors, 3,25-fold decreased expression of IDO1 was detected according to the tumor microenvironment (p=0,05), IDO1 expression was found to be more than 2 times higher in signet ring cell carcinoma (SRCC) and poorly differentiated tumors without distant organ metastasis (p<0,05), In GC, tryptophan level was found to be 1,6 times lower than in control (AUC:0889; cut off≤21,57; p<0001), Low tryptophan level was found in advanced tumor stage compared to early stage and in the presence of perineural invasion compared to its absence (p<0,05) The level of kynurenine was found to be approximately 1,8 times lower in SRCC (p=0,04), CONCLUSION: Increased tryptophan accumulation in the gastric tumor and its microenvironment, when catabolized via IDO1, exhibits histological type, tumor differentiation, and metastasis-promoting effects more prominently in aggressive subtypes such as SRCC.

The Role of Morphology in Predicting Fumarate Hydratase-deficient Uterine Leiomyomas in Young Women.

Hereditary leiomyomatosis and renal cell carcinoma is caused by germline mutations in the fumarate hydratase (FH) gene and is associated with an increased incidence of leiomyomas and a potentially aggressive variant of renal cell carcinoma. Pathologic evaluation of uterine leiomyoma can provide an opportunity for early recognition of the syndrome. We reviewed all archived slides of the cases to identify the characteristic morphologic features described for FH-deficient leiomyomas. We performed immunohistochemistry on whole sections of patients with uterine leiomyoma to evaluate for both FH and 2-succinocysteine (2SC) expression. Of the 106 cases, 19 showed the characteristic eosinophilic nucleoli with perinuclear halos, and 24 revealed a characteristic eosinophilic cytoplasmic inclusion consisting of pink globules present within the cytoplasm. Both of these morphologic findings were present together in 15 cases, and hemangiopericytomatous vessels were detected in 23 cases. The loss of FH protein expression was detected in 14 out of 106 cases (13%), and 13 out of 106 cases (12%) were positive for 2SC. We detected 10 cases with both 2SC-positive and FH expression loss. The presence of eosinophilic nucleoli with perinuclear halos and eosinophilic cytoplasmic inclusion was associated with both loss of FH protein expression and 2SC positivity ( P < 0.001). These findings underscore the importance of hematoxylin and eosin-based predictive morphology in FH-deficient uterine leiomyomas. Therefore, morphologic assessment of uterine leiomyomas for features of FH deficiency can serve as a screening tool for hereditary leiomyomatosis and renal cell carcinoma syndrome, allowing patients to be divided according to their hereditary risk assessment.

Ductal Carcinoma In Situ Arising in Sentinel Axillary Lymph Nodes Excised From Patients With Breast Carcinoma - A Potential Diagnostic Pitfall. Report of Two Cases.

We present two cases of ductal carcinoma in situ (DCIS) that arose in axillary lymph nodes excised as the sentinel lymph node from two patients with breast carcinoma. The patient ages were 72 and 36 years and both patients underwent mastectomy and axillary lymph node dissection. In addition to DCIS in the sentinel lymph node, the first patient had a wide DCIS and microinvasion in the ipsilateral breast and a micrometastasis in another sentinel lymph node. The second patient was operated on after neoadjuvant chemotherapy and had DCIS and a small focus of invasion, in addition to invasive and in situ ductal carcinoma in the lymph node having signs of chemotherapy-induced regression. The presence of DCIS was confirmed by use of the immunohistochemical method with antibodies against myoepithelial cells. As a potential source of cellular origin, DCIS was accompanied by benign epithelial cell clusters in the lymph node in both cases. Morphologic and immunohistochemical features were similar in breast and lymph node neoplasms. We conclude that DCIS may rarely develop from benign epithelial inclusions in the axillary lymph node and is a potential diagnostic pitfall in cases having ipsilateral breast carcinoma.

Evaluation of immune density, PD-L1, and CXCR4 expressions in metaplastic breast carcinoma to predict potential immunotherapy benefit.

Metaplastic breast carcinoma (MBC) -rare but fatal subtype of invasive breast carcinomas- provides limited benefit from conventional triple-negative breast carcinoma chemotherapy. We aimed to determine the immune density of this tumor and to evaluate of programmed death-ligand 1 (PD-L1) and chemokine receptor type 4 (CXCR4) expressions to determine whether it would benefit from immunotherapy. Clinicopathological characteristics of 85 patients diagnosed as MBC between 1997 and 2017 were retrospectively assessed. We evaluated the immunohistochemical expression of PD-L1 and CXCR4, and the extent of tumour infiltrating lymphocytes (TILs), with survival data. TILs groups were statistically significantly associated with lymph node status, histological subtype, squamous component, local recurrence and/or systemic metastasis, and disease-related deaths (p < 0.05). PD-L1 positivity in immune cells (ICs) has a statistically significant relationship with the presence of squamous component (p = 0.011) and HER2 positivity (p = 0.031). PD-L1 positivity in tumor cells (TCs) was found to be significantly more frequent in high-TILs density (p = 0.003). PD-L1 combined positive score was significantly associated with the tumors containing high-TILs density (p = 0.012) and squamous component (p = 0.035). Disease-free and disease-specific survival rates were found to be longer for the cases displaying PD-L1 positivity in ICs; and also PD-L1 positivity in ICs was found to be an independent prognostic factor. When the expression of CXCR4 was compared with clinicopathological and survival parameters, no statistically significant association was found (p > 0.05). Based on the results of this retrospective study, PD-L1 and TILs appear to be prognostic. This study provides rationale for further studies to determine whether a subset of patients with metaplastic breast cancer could derive a meaningful benefit from immune-targeting therapies.

Prognostic Implications of Immune Infiltrates in the Breast Cancer Microenvironment: The Role of Expressions of CTLA-4, PD-1, and LAG-3.

The assessment of immune infiltrate in invasive breast carcinomas (IBCs), most commonly referred to as tumor infiltrating lymphocytes (TILs), is gaining importance in the current quest for optimal biomarker selection and prediction of prognosis. In this study, the impact of intensity of TILs and expressions of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death-1 (PD-1), and lymphocyte activation gene 3 (LAG-3) in a group of breast carcinomas with regards to the prognosis and conventional pathologic parameters was scrutinized. For this purpose, 238 patients with IBCs containing different proportions of TILs were included in the study. IBCs with higher proportion of TILs were usually grade III carcinomas and correlated with poor prognostic features like receptor negativity, nonluminal intrinsic subtype (P<0.001). Similarly, PD-1 and LAG-3 positivity in immune cells (IC) were more likely to be positive in grade III IBC cases (P=0.004). In addition, PD-1 positivity in IC was more frequent in estrogen receptor-negative tumors (P=0.011) whereas LAG-3 positivity increased in large sized, estrogen receptor and progesterone receptor-negative tumors (P=0.050, 0.023, 0.04, respectively). CTLA-4 positivity in IC was more frequent in large-sized tumors (P=0.040). These 3 markers were also significantly associated with one another and also with the amount of TILs. In survival analysis, cases with prominent-TILs especially displaying CTLA-4, PD-1, and LAG-3 positivity appeared to have longer disease-free and overall survival (CTLA-4: P=0.027, P=0.024; PD-1: P=0.030, P=0.026; LAG-3: P=0.006, P=0.012, respectively). We conclude that the high proportion of TILs and as well as high expression of CTLA-4, PD-1, and LAG-3 in TILs have positively contributed to the outcome despite their correlation with poor conventional pathologic features. We suggest that these 3 immune markers can be used for the determination of proper treatment as well as prediction of prognosis in IBCs with TILs.

How accurate is 68Gallium-prostate specific membrane antigen positron emission tomography / computed tomography (68Ga-PSMA PET/CT) on primary lymph node staging before radical prostatectomy in intermediate and high risk prostate cancer? A study of patient- and lymph node- based analyses.

BACKGROUND AND AIM: Gallium-68 (68Ga)-Prostate Membrane Specific Antigen Positron Emission Tomography/Computed Tomography (68Ga-PSMA PET/CT) is an emerging diagnostic modality which is gaining importance in individualized prostate cancer (PCa) management era. This study aimed to investigate the diagnostic accuracy of 68Ga-PSMA PET/CT on primary LN staging before radical prostatectomy (RP) in intermediate and high risk PCa. MATERIALS AND METHODS: The retrospectively documented 49 patients with intermediate and high risk non-metastatic PCa who had 68Ga-PSMA PET/CT before RP were enrolled into this study. The histopathology of dissected LNs was used as reference standard to evaluate the accuracy of 68Ga-PSMA PET/CT on primary LN staging, both in per-patient (n = 49) and in per-node (n = 454) analyses. The diagnostic accuracy was investigated using sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), and by area under the curve (AUC) provided using receiver operating curve (ROC) analysis. RESULTS: Median age was 64 (48-79) years and, median and mean PSA values were 10 (1.31-138) ng/ml and 16.2 (±19.8) ng/ml, respectively. 22 (44.9%) and 27 (55.1%) of patients had intermediate and high risk PCa, respectively. A total of 5 (10.2%) patients had histopathologically proven LN metastasis and 3 (60%) of them was detected in 68Ga-PSMA PET/CT. In per patient analysis, the sensitivity, specifity, PPV and NPV of 68Ga-PSMA PET/CT on primary LN staging were 0.60, 0.96, 0.60 and 0.96, respectively. Among overall 454 LNs, 16 (3.5 %) of them were reported as metastatic in histopathology and, 13 (2.9%) of these metastatic LNs were detected in 68Ga-PSMA PET/CT. In per-node analysis, the sensitivity, specificity, PPV and NPV of 68Ga-PSMA PET/CT on primary LN staging were 0.82, 0.99, 0.87 and 0.99, respectively. The ROC analyses found AUCs for primary LN staging as 0.777 (95%CI:0.508-1.0) in per patient analysis and, as 0.904 (95%CI:0.790 - 1.0) in per node analysis, respectively. CONCLUSION: The use of 68Ga-PSMA PET/CT has promising diagnostic accuracy on primary LN staging before RP in intermediate and high risk PCa. However, the efforts should be taken to increase sensitivity of 68Ga-PSMA PET/CT in individualized treatment era.

DICER1 Mutations Occur in More Than One-Third of Follicular-Patterned Pediatric Papillary Thyroid Carcinomas and Correlate with a Low-Risk Disease and Female Gender Predilection.

Some pediatric papillary thyroid carcinoma (PPTC) cohorts have suggested a preliminary correlation with respect to DICER1 mutation status and histomorphology in both benign and malignant follicular cell-derived nodules; however, the data regarding correlates of DICER1-related sporadic PPTCs subtyped based on the 2022 WHO classification criteria are largely unavailable. The current study investigated the status of hotspot DICER1 mutations with clinical, histological and outcome features in a series of 56 patients with PPTCs with no clinical or family history of DICER1-related syndromic manifestation. Fifteen (27%) PPTCs harbored BRAF p.V600E. Eight (14%) cases of PPTCs harbored DICER1 mutations with no associated BRAF p.V600E. DICER1 mutations were identified in exons 26 and 27. A novel D1810del (c.5428_5430delGAT) mutation was also detected. We also confirmed the absence of hotspot DICER1 mutations in the matched non-tumor tissue DNA in all 8 DICER1-related PPTCs. The mean age of DICER1-harboring PPTCs was 15.1 (range: 9-18) years whereas the rest of this cohort had a mean age of 14.8 (range 6-18) years. With the exception of one PPTC, all DICER1-related PPTCs were seen in females (female-to-male ratio: 7). The female to male ratio was 3.8 in 48 DICER1-wild type PPTCs. In terms of histological correlates, 5 of 8 (63%) DICER1-mutant PPTCs were invasive encapsulated follicular variant papillary thyroid carcinomas (FVPTCs) including 4 minimally invasive FVPTCs and 1 encapsulated angioinvasive FVPTC, whereas the remaining 3 PPTCs were infiltrative classic papillary thyroid carcinomas (p < 0.05). The incidence of DICER1 mutations was 19.5% in BRAF p.V600E-wild type PPTCs. Sixty-three percent of DICER1 hotspot mutations occurred in invasive encapsulated FVPTCs, and this figure represents 38% of invasive encapsulated FVPTCs. Only one (12%) patient with DICER1-related disease showed a single lymph node with micro-metastasis. Unlike DICER1-wild type patients, no distant metastasis is identified in patients with DICER1-related PPTCs. The current series expands on the surgical epidemiology of somatic DICER1-related PPTCs by correlating the mutation status with the clinicopathological variables. Our findings underscore that female gender predilection and enrichment in low-risk follicular-patterned PTCs are characteristics of DICER1-related PPTCs.

Quantitative Proteomics Identifies Secreted Diagnostic Biomarkers as well as Tumor-Dependent Prognostic Targets for Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the third most common and most malignant urological cancer, with a 5-year survival rate of 10% for patients with advanced tumors. Here, we identified 10,160 unique proteins by in-depth quantitative proteomics, of which 955 proteins were significantly regulated between tumor and normal adjacent tissues. We verified four putatively secreted biomarker candidates, namely, PLOD2, FERMT3, SPARC, and SIRPα, as highly expressed proteins that are not affected by intratumor and intertumor heterogeneity. Moreover, SPARC displayed a significant increase in urine samples of patients with ccRCC, making it a promising marker for the detection of the disease in body fluids. Furthermore, based on molecular expression profiles, we propose a biomarker panel for the robust classification of ccRCC tumors into two main clusters, which significantly differed in patient outcome with an almost three times higher risk of death for cluster 1 tumors compared with cluster 2 tumors. Moreover, among the most significant clustering proteins, 13 were targets of repurposed inhibitory FDA-approved drugs. Our rigorous proteomics approach identified promising diagnostic and tumor-discriminative biomarker candidates which can serve as therapeutic targets for the treatment of ccRCC. IMPLICATIONS: Our in-depth quantitative proteomics analysis of ccRCC tissues identifies the putatively secreted protein SPARC as a promising urine biomarker and reveals two molecular tumor phenotypes.

Malignant Transformation of Parotid Gland Non-sebaceous Lymphadenoma: Case Report and Review of Literature.

Non-sebaceous lymphadenoma is a sporadic benign tumor of salivary glands. Histopathologic and immunohistochemical properties, diagnostic criteria, and theories for the histologic origin of the disease have been defined and well-discussed in the literature. However, none of the cases showed malignant transformation to date. We reported a case of 54 years old female patient with a right preauricular mass. Magnetic resonance imaging demonstrated a 2 cm, well-defined contrast-enhanced mass in the right parotid gland. Fine needle aspiration cytology was undiagnostic but suspicious for malignancy. Total parotidectomy with facial nerve preservation was done. In the histopathological examination, non-sebaceous lymphadenoma regions and malignant cells with abundant cytoplasm, large vesicular nuclei, and prominent nucleoli, which occupied approximately 70% of the mass, were seen. The diagnosis was undifferentiated carcinoma arisen from non-sebaceous lymphadenoma. Adjuvant radiotherapy was given. No recurrence was detected during ten months of follow-up. This case is the first case of a malignancy developed from non-sebaceous lymphadenoma.

Echogenic lymph nodes in the differential diagnosis of pediatric sarcoidosis.

We present a delayed diagnosis of sarcoidosis in an 11-year-old girl by demonstrating ultrasonographic imaging findings of granulomatous cervical and abdominal lymph node involvement. Pulmonary interstitial fibrosis in addition to multi-compartmental enlarged echogenic lymph nodes could be considered sarcoidosis. Punctate echogenic foci in the cervical lymph nodes should be considered in the differential diagnosis of sarcoidosis.