Pelvic lymph node dissection (PLND) is the most accurate procedure for lymph node (LN) staging in prostate cancer (PCa) patients. LN sectioning and hematoxylin and eosin (H&E) staining of at least one slice remains the gold standard for LN evaluation, potentially leading to misdetection of small metastatic focus. Entire LN analysis is possible with One-Step Nucleic Acid Amplification (OSNA) by detecting cytokeratin 19 (CK19) mRNA as a surrogate for LN invasion. This study aimed to compare postoperative performance of OSNA pooling with conventional H&E staining for pathological LN detection in PCa patients. POPCORN was an observational, prospective, and multicenter study of patients with PCa who underwent PLND. Dissected LNs were analyzed by both methods. This study included 2503 LNs from 131 patients, showing no statistically significant differences in pathological LN detection. Concordance between methods was high (93.9%), as were specificity (96.6%) and negative predictive value (96.6%) of OSNA pooling. The measure of agreement (Cohen’s Kappa [κ]) was 0.70. Only eight (6.1%) discordances were observed, including four misdetections from each method. Results showed a high concordance between OSNA pooling and H&E staining, suggesting that OSNA pooling may be a good alternative to H&E staining to detect LN metastases in PCa patients.

Dormant disseminated tumor cells (DTCs) remain viable for years to decades before establishing a clinically overt metastatic lesion. DTCs are known to be highly resilient and able to overcome the multiple biological hurdles imposed along the metastatic cascade. However, the specific metabolic adaptations of dormant DTCs remain to be elucidated. Here, we reveal that dormant DTCs upregulate de novo lipogenesis and favor the activation and incorporation of monounsaturated fatty acids (MUFAs) to their cellular membranes through the activation of acyl-coenzyme A synthetase long-chain family member 3 (ACSL3). Pharmacologic inhibition of de novo lipogenesis or genetic knockdown of ACSL3 results in lipid peroxidation and non-apoptotic cell death through ferroptosis. Clinically, ACSL3 was found to be overexpressed in quiescent DTCs in the lymph nodes of breast cancer patients and to significantly correlate with shorter disease-free and overall survival. Our work provides new insights into the molecular mechanisms enabling the survival of dormant DTCs and supports the use of de novo lipogenesis inhibitors to prevent breast cancer metastasis.

Supplementary Figure 2. Target efficiency of ATF4-siRNA, effects of ATF4 knockdown on tumor cell migration, and ATF4 expression in mammospheres.

Supplementary Table S2. Relapse-free survival of gene signature and single genes according to hormone status.

Non-tracked version of the supplementary information document that includes supplementary materials and methods, supplementary references and supplementary figure legends

In triple-negative breast cancer (TNBC), the pleiotropic NDRG1 (N-Myc downstream regulated gene 1) promotes progression and worse survival, yet contradictory results were documented, and the mechanisms remain unknown.Phosphorylation and localization could drive NDRG1 pleiotropy, nonetheless, their role in TNBC progression and clinical outcome was not investigated.We found enhanced p-NDRG1 (Thr346) by TGFβ1 and explored whether it drives NDRG1 pleiotropy and TNBC progression.In tissue microarrays of 81 TNBC patients, we identified that staining and localization of NDRG1 and p-NDRG1 (Thr346) are biomarkers and risk factors associated with shorter overall survival.We found that TGFβ1 leads NDRG1, downstream of GSK3β, and upstream of NF-κB, to differentially regulate migration, invasion, epithelial-mesenchymal transition, tumor initiation, and maintenance of different populations of cancer stem cells (CSCs), depending on the progression stage of tumor cells, and the combination of TGFβ and GSK3β inhibitors impaired CSCs.The present study revealed the striking importance to assess both total NDRG1 and p-NDRG1 (Thr346) positiveness and subcellular localization to evaluate patient prognosis and their stratification.NDRG1 pleiotropy is driven by TGFβ to differentially promote metastasis and/or maintenance of CSCs at different stages of tumor progression, which could be abrogated by the inhibition of TGFβ and GSK3β.

Supplementary Table S1. Overall survival in low- and high-expression cohorts.

Supplementary Figure 3. Screening of non-canonical molecular pathways and OncoPrint analysis of the eight-gene signature.

Supplementary Figure 1. Patient survival with high ATF4 expression in ER+ patients. ROC curve. Differential ATF4 expression induced by TGFβ1 and ERS. SBE motifs in ATF4 promoter and ChiP-Seq analysis.

The aim of the study was to stratify high-grade T1 (HGT1) bladder urothelial carcinoma into risk categories based on the presence of variant histology when compared to conventional urothelial carcinoma. The clinicopathological features of 104 HGT1 cases of urothelial carcinoma of the bladder with variant histology present in 34 (37%) were assessed. The endpoint of the study was disease-free survival and cancer-specific survival. Overall, variant histology was identified as a significant predictor of disease-free survival (P = 0.035). The presence of any specific variant histology (squamous, glandular, micropapillary, nested, microcystic, inverted growth, villous-like, basaloid, and lymphoepithelioma-like) was identified as a significant predictor of disease-free survival (P = 0.008) and cancer-specific survival (P = 0.0001) in HGT1 bladder cancer. Therefore, our results support including micropapillary HGT1 urothelial carcinoma within the aggressive high-risk category, as suggested by some recent clinical guidelines, but also favor nested, glandular, and basaloid to be placed in the high-risk category due to their potential of aggressive, life-threatening behavior and their limited response to bacillus Calmette-Guerin therapy. Conversely, the low-risk category would include urothelial carcinomas with squamous, inverted growth, or microcystic morphology, all with limited life-threatening potential and good response to current therapy. A very low-risk category would finally include patients whose tumors present villous-like or lymphoepithelioma-like morphology. In conclusion, our findings support the value of reporting the variant histology as a feature of variable aggressiveness in HGT1 urothelial carcinoma of the bladder.

Abstract Purpose: On the basis of the identified stress-independent cellular functions of activating transcription factor 4 (ATF4), we reported enhanced ATF4 levels in MCF10A cells treated with TGFβ1. ATF4 is overexpressed in patients with triple-negative breast cancer (TNBC), but its impact on patient survival and the underlying mechanisms remain unknown. We aimed to determine ATF4 effects on patients with breast cancer survival and TNBC aggressiveness, and the relationships between TGFβ and ATF4. Defining the signaling pathways may help us identify a cell signaling–tailored gene signature. Experimental Design: Patient survival data were determined by Kaplan–Meier analysis. Relationship between TGFβ and ATF4, their effects on aggressiveness (tumor proliferation, metastasis, and stemness), and the underlying pathways were analyzed in three TNBC cell lines and in vivo using patient-derived xenografts (PDX). Results: ATF4 overexpression correlated with TNBC patient survival decrease and a SMAD-dependent crosstalk between ATF4 and TGFβ was identified. ATF4 expression inhibition reduced migration, invasiveness, mammosphere-forming efficiency, proliferation, epithelial–mesenchymal transition, and antiapoptotic and stemness marker levels. In PDX models, ATF4 silencing decreased metastases, tumor growth, and relapse after chemotherapy. ATF4 was shown to be active downstream of SMAD2/3/4 and mTORC2, regulating TGFβ/SMAD and mTOR/RAC1–RHOA pathways independently of stress. We defined an eight-gene signature with prognostic potential, altered in 45% of 2,509 patients with breast cancer. Conclusions: ATF4 may represent a valuable prognostic biomarker and therapeutic target in patients with TNBC, and we identified a cell signaling pathway–based gene signature that may contribute to the development of combinatorial targeted therapies for breast cancer. Clin Cancer Res; 24(22); 5697–709. ©2018 AACR.

OBJECTIVE To report a clear cell renal cell carcinoma recipient of a metastasizing ductal carcinoma of the breast: A tumor-to-tumor metastasis. METHODS A 71 year-old woman with a past history of breast carcinoma, diagnosed 12 years before, underwent a nephrectomy for an incidental kidney mass found in a routine imaging examination. RESULTS Histological examination revealed foci of ductal carcinoma of the breast in an otherwise typical clear cell renal cell carcinoma of the kidney. Immunohistochemical examination confirmed a metastasis of an infiltrating breast carcinoma to a clear cell renal cell carcinoma (positive to GATA3, hormonal receptors and mamoglobin) in a clear cell renal cell carcinoma (positive to PAX8, CD10 and vimentin). CONCLUSIONS Awareness of this phenomenon should always be kept in mind by urologist in patients with a known history of a previous malignancy and by pathologists when finding a renal tumor with an unusual or dimorphic morphology. Immunohistochemistry plays an important role to establish the exact diagnosis.

High-grade papillary urothelial carcinoma with subepithelial connective tissue invasion (T1HG) is an aggressive disease at high risk of progression after transurethral resection/Bacillus Calmette-Guerin standardized therapy. The European Organization for Research and Treatment of Cancer has identified T1HG bladder carcinoma that is single and ≤3 cm in the largest dimension at first diagnosis as a category in which the prognosis cannot be further stratified based on conventional criteria. This category may benefit from biomarker analysis as a valuable tool to determine the patient's outcome. To further the issue of biomarkers in predicting aggressiveness in single T1HG bladder carcinoma ≤3 cm in greatest dimension at first diagnosis, we have conducted a validation study of the biomarker risk score set previously reported by our group. The study set included immunohistochemical detection of galectin-3, CD44, E-cadherin (E-CAD), CD138, p16, survivin, HYAL-1, and topoisomerase-IIα in 92 randomly selected specimens at participating institutions. Topoisomerase-IIα expression was identified as a predictor of disease-free survival. p16, survivin, and E-CAD expression predicted progression-free survival, but p16 and E-CAD also predicted overall survival. The current study validates a panel of immunohistochemical markers with the potential of being implemented in practice and supports the use of biomarkers in predicting aggressiveness in patients with first diagnosis of single T1HG bladder carcinoma ≤3 cm in greatest dimension and therefore in identifying patients who need closer surveillance or earlier aggressive treatment.

We present the clinicopathological features of 56 cases of the nested variant of urothelial bladder carcinoma. This is an uncommon variant of bladder cancer, recognized by the current WHO classification of urologic tumors. The nested component represented 100 % of the tumor in 24 cases. The architectural pattern of the tumor varied from solid expansile to infiltrative nests characterized by deceptively bland histologic features resembling von Brunn nests. Typical features of high-grade conventional urothelial carcinoma were present in 32 cases. Most neoplastic cells had nuclei of low to intermediate nuclear grade with occasional nuclear enlargement, most frequently seen in deep areas of tumor. The nested component expressed cytokeratins 7, 20, CAM5.2, and high molecular weight (34ßE12), p63, Ki67, p53, p27, and GATA3. Tumor extension was T1 (n = 9), minimally T2 (n = 10), T2a (n = 1), T2b (n = 4), T3a (n = 8), T3b (n = 13), and T4a (n = 11). On follow-up, 36 of patients died of or were alive with disease from 2 to 80 months (mean 21 months). Four patients died of other causes. Eleven other patients remained disease free. Univariate survival analysis showed no differences for nested carcinoma compared with conventional urothelial carcinoma. As in conventional urothelial carcinoma, in nested carcinoma of the bladder pT category defined different survival groups. In summary, nested variant of urothelial bladder carcinoma is typically associated with advanced stage. In samples of limited volume, it may be misdiagnosed as proliferation of von Brunn nests or other nested-like bladder lesions, delaying definitive therapy.

Since circulating tumor cells (CTCs) have metastatic potential, their genetic and phenotypic characteristics could provide crucial information to establish the most effective therapy. We assessed the clinical utility of a methodology that allows the simultaneous analysis of CTC phenotype and genotype in colon cancer patients and, in addition, whether this methodology could provide complementary information to that obtained by the primary tumor biopsy. Thirty-three non-metastatic (stages 0-III) colon cancer patients and 9 healthy donor samples were evaluated. All peripheral blood samples (10 ml) were analyzed by cytokeratin immunomagnetic enrichment. Eight samples were analyzed by immunocytochemistry and 25 samples were analyzed by FICTION technique for simultaneous cytokeratin expression and chromosome 17 and ERBB2 gene status. A further study was carried out in one patient who showed CTC heterogeneity in chromosomal abnormalities. We analyzed HER2 protein expression on CTCs and FISH and HER2 protein expression in primary tumor of this patient. Our results show that 9.09% of patients had cytokeratin-positive CTCs (CK+/CTCs in peripheral blood). One of the patients showed heterogeneity in chromosomal 17 abnormalities and two different CK expression patterns on CTCs: one CK+/CTCs and one CK-/CTCs. Furthermore, 63.33% of these CTCs overexpressed HER2 protein while the primary tumor of this patient was diploid and did not express HER2 protein. We describe a methodology that allows the simultaneous genetic and phenotypic analysis of CTCs in colon cancer patients, which may provide essential information to select patients who might benefit from specific therapy.

To report two cases of late metastases of clear cell renal cell carcinoma.Two patients, a male and a female with history of nephrectomy 17 and 16 years before for renal cell carcinomas, presented new tumours in the thyroid and pancreas, which were excised.Pathology reported that both lesions were clear cell tumours and immunohistochemically they were consistent with metastases from clear renal cell carcinomas.1) Previous history of any type of carcinoma should suggest the possibility of metastases when facing a thyroid or pancreatic nodule. 2) All-life follow-up should be made, nephrectomy (resection) for a renal cell carcinoma. 3) In the presence of a clear cell tumour of the thyroid or pan-creatic glands, the differential diagnosis must always include metastatic renal cell carcinoma. 4) The treatment of choice is surgical resection.

To report two cases of late metastases of clear cell renal cell carcinoma.Two patients, a male and a female with history of nephrectomy 17 and 16 years before for renal cell carcinomas, presented new tumours in the thyroid and pancreas, which were excised.Pathology reported that both lesions were clear cell tumours and immunohistochemically they were consistent with metastases from clear renal cell carcinomas.1) Previous history of any type of carcinoma should suggest the possibility of metastases when facing a thyroid or pancreatic nodule. 2) All-life follow-up should be made, nephrectomy (resection) for a renal cell carcinoma. 3) In the presence of a clear cell tumour of the thyroid or pan-creatic glands, the differential diagnosis must always include metastatic renal cell carcinoma. 4) The treatment of choice is surgical resection.