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Supplemental Methods 1. Exome sequencing pipeline Supplemental Methods 2. RNA seq pipeline Supplemental Methods 3. DNA methylation profiling pipeline Supplemental Methods 4. Fluorescence in situ hybridization Supplemental Methods 5. TCGA samples with possible copy neutral loss of heterozygosity in 3p21 and 3p25 regions

Purpose: Sarcomatoid renal cell carcinoma (SRCC) ranks among the most aggressive clinicopathologic phenotypes of RCC. However, the paucity of high-quality, genome-wide molecular examinations of SRCC has hindered our understanding of this entity.Experimental Design: We interrogated the mutational, copy number, and transcriptional characteristics of SRCC and compared these data with those of nonsarcomatoid RCC (RCC). We evaluated whole-exome sequencing, single-nucleotide polymorphism, and RNA sequencing data from patients with SRCC (n = 65) and RCC (n = 598) across different parent RCC subtypes, including clear-cell RCC, papillary RCC, and chromophobe RCC subtypes.Results: SRCC was molecularly discrete from RCC and clustered according to its parent RCC subtype, though with upregulation of TGFβ signaling across all subtypes. The epithelioid (E-) and spindled (S-) histologic components of SRCC did not show differences in mutational load among cancer-related genes despite a higher mutational burden in S-. Notably, sarcomatoid clear-cell RCC (SccRCC) showed significantly fewer deletions at 3p21-25, a lower rate of two-hit loss for VHL and PBRM1, and more mutations in PTEN, TP53, and RELN compared with ccRCC. A two-hit loss involving VHL predicted for ccRCC and a better prognosis, whereas mutations in PTEN, TP53, or RELN predicted for SccRCC and worse prognosis.Conclusions: SRCC segregates by parent subtype, and SccRCC has a fundamentally different early molecular pathogenesis, usually lacking the classic 3p21-25 deletion and showing distinctive mutational and transcriptional profiles. These features prompt a more precise molecular classification of RCC, with diagnostic, prognostic, and therapeutic implications. Clin Cancer Res; 23(21); 6686–96. ©2017 AACR.See related commentary by Bergerot et al., p. 6381

Figure S1. Biphasic histologic components of sarcomatoid renal cell carcinoma. The macrodissected paired epithelioid or carcinomatous (E) and spindled or sarcomatoid (S) components of clear cell RCC (upper panel), Papillary RCC (middle panel), and chromophobe RCC (lower panel). H&E stain, scale bar 200 �m. Figure S2. Sarcomatoid ccRCC shows fewer VHL deletions. (A) Clear cell RCC (H&E stain, scale bar 100 µm) with (B) Fluorescence in situ hybridization (FISH) image showing paired CEN3q signals (green) and a single VHL signal (red). (C) Sarcomatoid ccRCC (H&E stain, scale bar 100 µm) with (D) FISH image showing balanced CEN3q (green) and VHL (red) signals. (E) Box plot showing significantly higher VHL/3q ratios associated with sarcomatoid histology, P

Table S7a. Ingenuity Pathway Analysis of SRCC vs RCC Table S7b. Ingenuity Pathway Analysis of SccRCC vs advanced stage ccRCC Table S7c. Ingenuity Pathway Analysis of E vs S components of ccRCC

Clear-cell renal cell cancer (CRCC) is initiated typically by loss of the tumor-suppressor VHL, driving constitutive activation of hypoxia-inducible factor-1 (HIF1) and HIF2. However, whereas HIF1 has a tumor-suppressor role, HIF2 plays a distinct role in driving CRCC. In this study, we show that the HIF1α E3 ligase hypoxia-associated factor (HAF) complexes with HIF2α at DNA to promote HIF2-dependent transcription through a mechanism relying upon HAF SUMOylation. HAF SUMOylation was induced by hypoxia, whereas HAF-mediated HIF1α degradation was SUMOylation independent. HAF overexpression in mice increased CRCC growth and metastasis. Clinically, HAF overexpression was associated with poor prognosis. Taken together, our results show that HAF is a specific mediator of HIF2 activation that is critical for CRCC development and morbidity. Cancer Res; 75(2); 316–29. ©2014 AACR.

Supplemental methods. A, B) Sequences for the A) P117 Luc, and B) P117 HRE reporters containing the HAF binding site, the hypoxia responsive element (HRE) and the HIF ancillary sequence (HAS), with the restriction enzyme sites used for ligation into the pGL4.17 construct. C) PCR primers used in ChIP assays. Supplementary data S1. A) Quantitative TaqMan RT-PCR showing the effect of transient HAF overexpression on the transcription of HIF-2 target genes in pVHL-deficient A498 CRCC cells B) Quantitative TaqMan RT-PCR showing the effect of transient HAF overexpression on the transcription of OCT-3/4. SOX2 and NANOG in normoxia and 24hours' hypoxia in A498 CRCC cells. Supplementary data S2. A) Effects of HAF overexpression on luciferase activity of co-transfected P117 Luc or P117 containing a deletion of the HAF site (P117D), in PANC1 cells in normoxia. B) Western blot showing knockdown specificity of siRNAs targeting HIF-2α (siH2_1, siH2_2) or HAF (siHAF_1, siHAF_2) when transfected with the P117 reporter in 786-0 cells. Supplementary data S3. A) Effect of hypoxia on SUMO-2/3 modification of HAF in 786-0 cells. B) Effect of hypoxia on SUMO-1 modification of HAF in PANC-1 cells. C) Western blot showing immunoprecipitation of HIF-2α from lysates of PANC-1 cells. Supplementary data S4. A-B) Immunocytochemistry confirming specificities of A) HAF, and B) HIF-2α antibodies. Supplementary data S5. A) Growth curve of HAF overexpressing 786-0 cells in normoxia. B) Western blot showing GFP expression in suspected lesions observed in the GI tracts of mice bearing renal orthotopic 786-0 HAF or vector expressing CRCC cells. Supplementary data S6. A) Table showing multiple disease parameters of the patients represented on the CRCC TMA. B) Scatterplot showing correlation between nuclear and cytoplasmic HAF localization in CRCC TMA. Supplementary data S7. Kaplan-Meier curves showing relationship between good versus intermediate Motzer criteria in grade III/IV CRCC. Supplementary data S8. A-B) Kaplan-Meier curves showing relationships between levels of A) HIF-2α or B) HIF-1α and progression free survival (PFS) in grade III/IV CRCC.

This work starts presenting recursive equations in the domain of sequences indexed by integers and their solutions obtained by applying the successive operations introduced in Moesia (2017). Following the original approach, the methodology is developed considering the traditional algebraic group structure. Starting from the dual interpretation of recursions, the notion of successive operations applied on sequences indexed by integers is revisited and some properties highlighted. More important, the successive operations approach is a general way to study previously extended functions from N to Z. As a demonstration of this capability, a class of binomial numbers extensions from the natural domain to the integer one is established here, allowing seeing that different pre-existing extensions of binomial numbers are, in fact, particular cases of just one general extension, developed in this work through the successive product machinery. To exemplify, binomials derived from two known factorial extensions are represented in this framework and their most known recursive expressions are studied. From this study it is also verified that by using the successive product it is possible to avoid extensive demonstrations required by standard methods, resulting in remarkably short and simple proofs.

Objectives: The objectives of the study were to assess the technical success, efficacy, and complications of the combination of transarterial embolization (TAE) followed by percutaneous ablation in treating stage cT1b and central renal tumors. Material and Methods: An institutional registry was reviewed for patients who underwent TAE followed by percutaneous computed tomography (CT)-guided renal ablation from 2007 to 2018. Twenty-eight consecutive patients (median age 69 years; range 45–89 years) with tumor size ranging from 25 to 58 mm (average 45 mm), including 24 patients with T1b tumors and four with central T1a tumors, were identified. Nineteen patients had TAE followed by cryoablation (CA), and nine patients had TAE followed by radiofrequency ablation (RFA). Technical success, local tumor control, and post-procedure complications were retrospectively reviewed. Results: All procedures were technically successful. Local tumor control was achieved in 26 of 28 patients (92.9%) at a median follow-up of 26 months. Local tumor recurrence was found in two of 28 patients, with a median time to local recurrence of 15 months. The recurrences occurred in one patient who had TAE plus CA and in one patient who had TAE plus RFA. Self-limited hematoma and hematuria were minor complications observed in 3 patients (10.7%). Only one patient had a major complication. This patient developed ureteral obstruction and perirenal abscess requiring ureteral stent and percutaneous drainage without long-term sequela. Conclusion: In this cohort of patients, a combination of TAE and percutaneous CT-guided ablation was an effective, well-tolerated, and safe treatment for patients with T1b and central renal tumors.

To evaluate the impact of microscopically positive vascular margins on recurrence and cancer-specific survival (CSS) in patients with renal cell carcinoma (RCC) with venous thrombusWe reviewed the records from the period 1993 to 2009 of consecutive patients treated surgically for RCC with venous tumour thrombus at the University of Texas MD Anderson Cancer Center. Patients with metastatic disease, positive soft tissue margins or gross residual disease at time of thrombectomy were excluded. The primary outcome measures were local or systemic disease recurrence, and CSS. Univariate and multivariate analysis were used to evaluate whether microscopically positive vascular margins were associated with RCC recurrence or CSS after nephrectomy with thrombectomy.A total of 256 patients with RCC were identified with a median (interquartile range) follow-up of 36.7 (18.4-63.5) months. Microscopic tumour was present at the margin of resection in 47 patients (18.4%). The median recurrence-free interval was significantly shorter in patients with positive vascular margins: 22.1 vs 70.2 months (P = 0.009). The rate of local recurrence was higher in patients with positive vein margins: 12.8 vs 4.3% (P0.01). Local recurrence without concomitant systemic recurrence was identified in only two of 256 (0.8%) patients. Patients with positive vascular margins had significantly worse CSS times compared with patients with negative vascular margins: 37.7 vs 93.0 months (P = 0.004). In multivariable analysis, positive vascular margins were found to be independently predictive of local recurrence but not of systemic recurrence or CSS.Complete surgical excision should always be attempted because positive vascular wall margins increase local recurrence rates. Invasion of RCC into the vein wall at the resection margin is associated with aggressive tumour biology, and the majority of patients with positive vascular wall margins experience systemic recurrence.

In this work we discuss stable equal-order finite element formulations for incompressible flow problems based on Petrov–Galerkin methods, constructed by adding to the classical Galerkin formulation least-squares of the governing equations. Continuous and discontinuous pressure interpolations are considered. Numerical results are presented reinforcing the numerical analysis.