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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

Introduction: Next Generation Sequencing, or NGS technology is gaining acceptance in diagnostic laboratories for multigene panel testing. However questions remain about the sensitivity, specificity and clinical implications of this new technology and the expanded testing it enables. Moreover, questions are raised as to whether new laboratories using these methods, typically without the benefit of large historical proprietary databases, can provide similar assessments of pathogenicity as do the previously established providers. Expanding on our recently published work (Kurian et al., JCO 2014) we considered whether NGS testing in an independent laboratory can replace traditional BRCA1/2 tests in patients indicated for hereditary breast/ovarian cancer testing. Methods: We recruited over 800 patients who were indicated for BRCA1/BRCA2 testing under clinical management guidelines, and collected over 200 additional samples to increase the power of this ongoing study. All were tested using an NGS-based multigene panel which reported both DNA sequence and copy-number alterations for BRCA1/2 and 27 other known cancer risk genes. Traditional genetic testing results using established technologies were also available for comparison. In this report we focus on the results for BRCA1/2 and 27 other known cancer risk genes. Results: Sensitivity was high: 261 alterations (196 pathogenic and 65 others) were reported in the traditional genetic data, and all were detected by NGS when the corresponding test was available. In this set are 141 alterations considered technically challenging for NGS: insertions, deletions, and complex sequence changes, as well as very large (chromosome) and small (single exon) copy number changes. Specificity was also high: all NGS variants for which we sought confirmation using independent methods (n>2000) were confirmed, including 51 alterations not previously reported. Determination of pathogenicity was also highly concordant: in all but 2 cases positive reports agreed, and in these 2 cases data were available in the literature to support pathogenicity, although not at a level which meets recent guidelines from the American College of Medical Genetics (ACMG). It is not clear from the diagnostic reports exactly what evidence supported pathogenicity in the traditional data for these 2 cases. Rates of Variants of Unknown Significance (VUS) in BRCA1/2 were somewhat different: about 7% of cases in the NGS data vs. about 4% of fully tested cases in the traditional data had an uncertain report. The root of this difference is also unclear as details are not provided in the traditional reports. Conclusions: NGS can be a viable replacement for traditional genetic testing for hereditary breast and ovarian cancer. Interpretation concordance is high but fully evaluating the details of this is hampered by the limited reporting of proprietary data by some established laboratories. Recent efforts to establish large public databases of genetic information (particularly ClinVar) will promote greater transparency and accountability and thus can help improve access to high quality care for hereditary conditions. Note: All of the variants in this study and their interpretations will be released to public databases by the time of the meeting. Citation Format: Stephen Lincoln, Allison Kurian, Andrea Desmond, Geoffrey Nilsen, Kevin Jacobs, Shan Yang, Reece Hart, Federico Monzon, Leif Ellisen, James Ford. Technical evaluation of multigene testing for hereditary breast and ovarian cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-12-07.

Since their discovery, microRNAs (miRNAs)20 have shown great promise in a wide array of clinical applications. In some cases, the use of miRNAs as new diagnostic markers might help answer diagnostic dilemmas that gene expression analyses or other types of analyses have not been able to satisfactorily address. In other instances, it is easy to perceive certain miRNAs as targets for novel therapies that downregulate an entire pathway via the targeting of a single miRNA. When and if this concept will come to fruition remain unclear. Scientists agree that this field of biology is exciting, offers much promise, and has numerous advantages, compared with experiences with other biomolecules. In this Q&A, 5 individuals with extensive miRNA experience share their vision for where the miRNA field is heading and whether we, in the clinical laboratory, will experience its implications soon. What are the most important characteristics of miRNAs that increase their potential as novel diagnostic or therapeutic targets? George Calin: One important characteristic of miRNAs that makes them an exciting potential biomarker for diagnosis and/or a target for therapy is the fact that specific miRNAs target multiple components from the same pathway. For example, the miR-15a/16–1 cluster targets genes from the apoptotic pathway: BCL2 21 (B-cell CLL/lymphoma 2) and MCL1 [myeloid cell leukemia sequence 1 ( BCL2 -related)]. Any given miRNA can regulate numerous genes, and each gene can be regulated by different miRNAs. Pierre Cordelier: In cancer research, miRNAs can differentiate normal from cancerous tissues and, more importantly, can discriminate different subtypes of cancer. The high stability of miRNAs in tissues and fluids is another key advantage that increases their potential as diagnostic markers over messenger RNA (mRNA). In addition, they can be quantified in very low amounts of material and in highly degraded samples. This is of prime importance …

Alpha-methyl CoA racemase (AMACR) is overexpressed in several human cancers, most notably colon and prostate. AMACR expression in the prostate has been investigated primarily in patients, in an older age group, treated for prostatic carcinoma and benign prostatic hypertrophy. No studies have assessed the age distribution of AMACR expression in normal men. Archival paraffin-embedded prostate tissue from 41 organ donor men (age range, 13-63 years) with no evidence of prostate neoplasia was stained with a monoclonal antibody for AMACR. Intensity was graded on a scale of 0 to 3. Semi-quantitative analysis of staining in acinar cells was used to generate a composite score (CS) [Sigma(% area x intensity)] for each case. Nondonor cases with foci of prostate cancer and high-grade prostatic intraepithelial neoplasia (PIN) were used as external positive controls for AMACR. These sections were also stained for Ki-67, to assess proliferative index. The 41 cases encompassed different age groups (13-20 years, N = 11; 20-45 years, N = 17; >45 years, N = 13). Acinar cells showed granular cytoplasmic staining. Focal positive staining was also seen in the prostatic urethra and the periurethral glands. There was wide variation in the level of expression within each age group. The level of expression seen in subjects younger than 45 years was higher (mean CS = 41.3; median CS = 22.5) than that seen in subjects older than 45 years (mean CS = 8.8; median CS = 9.0) with a P value of 0.01. Most cases in the control set of prostatic adenocarcinoma cases showed moderate to strong staining. A negative correlation was seen evaluating CS and age in subjects 20 years of age and older (r = -0.47). Ki-67 staining was variable. 1) AMACR expression can be seen in benign prostatic glandular epithelium, across all age groups. However, it is age-related, with significantly lower expression in subjects younger than 45 years. This could account for the negative staining reported in benign glands, due to biased sampling of the older population. 2) Focal positive staining is seen in the prostatic urethra and periurethral glands in 71% of the cases, with no age correlation. This is of concern because this epithelium could potentially be misinterpreted as foci of PIN. 3) The low expression of AMACR in benign glands in the older age group makes this marker useful in detecting malignancy. However, AMACR staining should be interpreted with caution and the diagnosis of PIN or prostate cancer should be rendered only with convincing histologic evidence. 4) Ki-67 staining was very variable and showed no correlation with age and AMACR expression levels. AMACR expression had no correlation with proliferative index.

Objective: To assess the effects of hydrosalpinx fluid on human cytotrophoblast viability and function in vitro. Design: Human cytotrophoblasts obtained from third-trimester placentas were cultured in vitro with hydrosalpinx fluid, and cell viability and protein production were assayed. Setting: A university hospital. Patient(s): Ten hydrosalpinx fluid samples obtained from seven women with clearly diagnosed hydrosalpinges. Intervention(s): Recovery of hydrosalpinx fluid by transvaginal aspiration or at the time of surgery. Main Outcome Measure(s): Cell viability was assessed by the XTT assay. Secretion of trophoblast oncofetal fibronectin (tropho-uteronectin) and β -hCG by cultured trophoblasts was determined by Western blot and ELISA of the culture media. Result(s): With increasing concentrations of hydrosalpinx fluid from 0% to 20%, there was a significant increase in trophoblast cell viability (1.63-fold increase in 20% hydrosalpinx fluid). Likewise, both Western blot and ELISA assays demonstrated a significant increase in trophouteronectin production by trophoblasts with increasing hydrosalpinx fluid concentrations (3.76-fold increase in 20% hydrosalpinx fluid). β -Human chorionic gonadotropin production also increased significantly in the presence of hydrosalpinx fluid (3.31-fold increase in 20% hydrosalpinx fluid). Conclusion(s): These findings suggest that hydrosalpinx fluid improves human trophoblast viability in vitro and enhances the production of tropho-uteronectin and β -hCG by these cells.

OBJECTIVE: Human trophoblast proteolytic activity is believed to have implications for early implantation events and maintenance of chorionic structural integrity later in gestation. Abnormal release of chorion-derived extracellular matrix proteins such as fibronectin may identify patients at risk for preterm labor and delivery. The aim of this study was to characterize the enzyme(s) potentially responsible for trophoblast-mediated proteolysis of fibronectin. STUDY DESIGN: Human term cytotrophoblasts were analyzed for their capacity to cleave fibronectin into discrete proteolytic fragments. Selective protease inhibitors were used to characterize trophoblast-derived enzymes with fibronectinase activity. Analysis and quantitation of fibronectin fragment release was determined by Western immunoblots and enzyme-linked immunosorbent assays. RESULTS: Fibronectinase activity in trophoblast cultures was found to be both cell mediated and secreted, with the release of discrete fibronectin fragments into the media. Cell-mediated proteolytic activity could be partially inhibited by serum, whereas conditioned media containing fibronectinase activity was completely inhibited by serum, a serine protease inhibitor, and a selective inhibitor of urokinase-type plasminogen activator. Digestion of fibronectin with pure urokinase produced a similar pattern of fibronectin fragments compared with fibronectinase-generated fragments. Immunodepletion of urokinase from trophoblast media abolished fibronectinase activity. CONCLUSIONS: Trophoblast-derived urokinase-type plasminogen activator has significant proteolytic activity in vitro with the capability of cleaving fibronectin into discrete fragments. In early pregnancy this activity could be part of the enzymatic cascade leading to uterine extracellular matrix remodeling and implantation. Later in pregnancy trophoblast-derived urokinase could promote normal or inflammation-induced changes in the chorionic extracellular matrix. (Am J Obstet Gynecol 1997;176:58-65.)

A case of locally invasive, long-standing desmoplastic and amelanotic malignant melanoma is described in an 84-year-old man. Histologic examination of the involved periorbital tissue showed neoplastic foci exhibiting a novel pattern reminiscent of microvascular proliferation. These regions were characterized by malignant, S-100-positive tumor cells lining vessel-like spaces in transverse sections and forming tubuler-like structures in longitudinal sections. Recent data indicate that melanoma cells may express genes and patterns of differentiation in vitro akin to endothelial cells. Because angiosarcoma often involves facial and scalp skin of elderly individuals, awareness of angiomatoid differentiation in melanoma has important diagnostic implications. HUM PATHOL 31:1520-1522.

Objective: The rarer allele of a polymorphism within the promoter region at position –308 of the gene for tumor necrosis factor α is associated with increased gene transcription. In this study we tested the hypothesis that this rarer allele is associated with spontaneous preterm birth. Study Design: We conducted a case-control study of women admitted to our labor and delivery unit. To assess data from a single racial group with a high incidence of preterm birth we restricted our analysis to African American women, who contributed 73.6% of the samples collected during the study period. Case patients (n = 55) were defined as women who were delivered before 37 weeks' gestation after idiopathic preterm labor or preterm premature rupture of membranes. Control subjects (n = 110) included women who were delivered after 37 weeks' gestation and had no history of preterm delivery. We also performed subgroup analyses of women with idiopathic preterm labor and delivery (n = 29) and women who were delivered preterm after preterm premature rupture of the fetal membranes (n = 26). Results: Although carriers (homozygotes plus heterozygotes) of the rarer allele of the polymorphism at position –308 in the gene for tumor necrosis factor α were not significantly more common among women who were delivered preterm (n = 24/55, 44%) than among control subjects (n = 33/110, 30%, P = .08, odds ratio 1.81, 95% confidence interval 0.92-3.54), carriers of the rarer allele were more common among women who were delivered preterm after preterm premature rupture of membranes (n = 15/26, 58%) than among control subjects ( P = .008, odds ratio 3.18, 95% confidence interval 1.33-7.83). Conclusions: Our results demonstrate an association between allelic variants of the polymorphism at position –308 in the gene for tumor necrosis factor α and preterm birth after preterm premature rupture of the fetal membranes. We hypothesize that host susceptibility to environmental factors, such as hyperresponsiveness of the gene for tumor necrosis factor α to genital tract infection, may promote preterm premature rupture of the fetal membranes and subsequent preterm delivery. (Am J Obstet Gynecol 1999;180:1297-302.)

OBJECTIVE: Oncofetal fibronectin reactive with antibody FDC-6 has been associated with trophoblastic implantation and chorion structural stability. Abnormal release of this fibronectin into cervical and vaginal secretions has identified patients at risk for preterm labor and delivery. The aim of this study was to determine whether trophoblast-oncofetal fibronectin contains other novel epitopes distinct from the FDC-6 binding site. STUDY DESIGN: Antitrophoblast fibronectin hybridomas were generated and screened by comparative immunoassays. One specific monoclonal antibody, X18A4, was identified and compared with antibody FDC-6 by immunocytochemical and immunoblot analyses. Both antibodies were also evaluated in "sandwich"-type double monoclonal immunosorbent assays. RESULTS: X18A4 and FDC-6 bind avidly and noncompetitively to distinct epitopes within oncofetal fibronectin. They exhibit similar immunohistochemical staining of the extracellular matrix within placental tissue, ovarian epithelial tumors, and cultured trophoblasts. However, in contrast to FDC-6, X18A4 has no detectable binding activity to human plasma fibronectin, and its binding to oncofetal fibronectin was unaffected by enzymatic deglycosylation. Immunoblot analyses of oncofetal fibronectin proteolytic digests suggest that X18A4 binds near or within the alternatively spliced type III connecting segment domain. CONCLUSIONS: X18A4 identifies and binds with high affinity to a new epitope within oncofetal fibronectin, distinct from the FDC-6 binding site. Because X184A displays no detectable binding to plasma fibronectin, it could be used as an important adjunctive antibody for enhancing the specificity of clinically based oncofetal fibronectin diagnostic assays.