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Chromophobe renal cell carcinoma (chRCC) accounts for approximately 5% of all renal cancers and around 30% of chRCC cases have mutations in TP53. chRCC is poorly supported by microvessels and has markably lower glucose uptake than clear cell RCC and papillary RCC. Currently, the metabolic status and mechanisms by which this tumor adapts to nutrient-poor microenvironments remain to be investigated. In this study, we performed proteome and metabolome profiling of chRCC tumors and adjacent kidney tissues and identified major metabolic alterations in chRCC tumors, including the classical Warburg effect, the downregulation of gluconeogenesis and amino acid metabolism, and the upregulation of protein degradation and endocytosis. chRCC cells depended on extracellular macromolecules as an amino acid source by activating endocytosis to sustain cell proliferation and survival. Inhibition of the phospholipase C gamma 2 (PLCG2)/inositol 1,4,5-trisphosphate (IP3)/Ca2+/protein kinase C (PKC) pathway significantly impaired the activation of endocytosis for amino acid uptakes into chRCC cells. In chRCC, whole-exome sequencing revealed that TP53 mutations were not related to expression of PLCG2 and activation of endocytosis. Our study provides novel perspectives on metabolic rewiring in chRCC and identifies the PLCG2/IP3/Ca2+/PKC axis as a potential therapeutic target in patients with chRCC. Significance: This study reveals macropinocytosis as an important process utilized by chRCC to gain extracellular nutrients in a p53-independent manner.

Chromophobe renal cell carcinoma (chRCC) and renal oncocytoma are closely related, rare kidney tumors. Mutations in complex I (CI)-encoding genes play an important role in dysfunction of the oxidative phosphorylation (OXPHOS) system in renal oncocytoma, but are less frequently observed in chRCC. As such, the relevance of OXPHOS status and role of CI mutations in chRCC remain unknown. To address this issue, we performed proteome and metabolome profiling as well as mitochondrial whole-exome sequencing to detect mitochondrial alterations in chRCC tissue specimens. Multiomic analysis revealed downregulation of electron transport chain (ETC) components in chRCC that differed from the expression profile in renal oncocytoma. A decrease in mitochondrial (mt)DNA content, rather than CI mutations, was the main cause for reduced OXPHOS in chRCC. There was a negative correlation between protein and transcript levels of nuclear DNA- but not mtDNA-encoded ETC complex subunits in chRCC. In addition, the reactive oxygen species scavenger glutathione (GSH) was upregulated in chRCC due to decreased expression of proteins involved in GSH degradation. These results demonstrate that distinct mechanisms of OXPHOS exist in chRCC and renal oncocytoma and that expression levels of ETC complex subunits can serve as a diagnostic marker for this rare malignancy. Significance: These findings establish potential diagnostic markers to distinguish malignant chRCC from its highly similar but benign counterpart, renal oncocytoma.

Recent next-generation sequencing studies of clear cell renal cell carcinoma (ccRCC) have identified point mutations in chromatin-modifying genes, among them PBRM1 which is now the second most frequently mutated gene in RCCs (in up to 41% ccRCCs cases) and belongs to the m1 TCGA (The Cancer Genome Atlas) subset. The occasional presence of PBRM1 mutations in the absence of VHL mutations indicates, that PBRM1 might be another driver tumor suppressor gene in ccRCCs. We were interested in examining PBRM1 expression at mRNA and protein levels and correlated these findings to histopathological features of RCC patients. Relative gene expression (RGE) data were obtained by Q-PCR from matched tumor and adjacent normal fresh frozen ccRCC tissues from 57 patients who underwent radical nephrectomy at our clinic. Papillary (pRCC) as well as chromophobic cases (chRCC) were added to analyze the expression of splice variants of PBRM1. Comparative Western blot analyses as well as semi-quantitative immuno-histochemical staining (IHC) for PBRM1 and VHL were performed on all ccRCC cases. In 78.9% of cases (45/57) PBRM1 mRNA was downregulated at least 1.5-fold, 7% (4/57) showed PBRM1 upregulation, and 14% (8/57) displayed no obvious expression changes between tumor and corresponding tumor tissue. Interestingly, 21 of 45 ccRCCs tumor tissues expressed splice variant 4 more abundantly when compared to normal tissue, whereas the normal tissue preferentially expressed splice variant 1 that includes an additional exon. Remarkably, this differential expression picture is completely reversed in pRCC and chRCC cases. The majority of 57 ccRCCs displayed weak nuclear PBRM1 staining (52.6%), whereas 31.6% showed moderate and 15.8% strong staining. However, we were not able to demonstrate a significant correlation of IHC expression levels, neither to tumor staging nor to Fuhrman grading. The observed high frequency of decreased expression of the chromatin-remodeling gene PBRM1 on mRNA (78.9%) and protein levels (52.6%), respectively, as wells as a high mutation rate (about 15% in our data set) indicate a substantial role of PBRM1 in the tumorigenesis of ccRCCs. Preferences in the expression of different PBRM1 splice variants warrant further investigation with regard to renal cell carcinoma development. Citation Format: Hans Krause, Anica Högner, Burkhard Jandrig, Ergin Kilic, Carsten Kempkensteffen. Expression changes of the chromatin modifier PBRM1 in human renal cell carcinomas in relation to histopathological features. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3181.

Here, we show that carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed in umbrella cells of bladder urothelium but is down-regulated in superficial bladder cancer, such as histologic tumor stage a (pTa) and transitional cell carcinoma in situ (pTis). Concurrently, CEACAM1 is up-regulated in the endothelia of adjacent angiogenic blood vessels. Mimicking the CEACAM1 down-regulation in the urothelium, CEACAM1 was silenced in bladder cancer cell lines 486p and RT4 using the small interfering RNA technique. CEACAM1 down-regulation was confirmed at the protein level by Western blot analyses. CEACAM1 silencing leads to a significant up-regulation of vascular endothelial growth factor (VEGF)-C and VEGF-D in quantitative reverse transcription-PCR. Correspondingly, supernatants from the CEACAM1-overexpressing bladder cancer cell lines reduce, but those from CEACAM1 silencing induce endothelial tube formation and potentiate the morphogenetic effects of VEGF. These data suggest that the epithelial down-regulation of CEACAM1 induces angiogenesis via increased expression of VEGF-C and VEGF-D. Inversely, CEACAM1 is up-regulated in endothelial cells of angiogenic blood vessels. This in turn is involved in the switch from noninvasive and nonvascularized to invasive and vascularized bladder cancer. CEACAM1 appears to be a promising endothelial target for bladder cancer therapy.

Background: The FGFR1-gene (fibroblast growth factor receptor 1) is located at chromosome 8p12 encoding for a tyrosine kinase. FGFR1 is involved in cell cycle, differentiation, survival, apoptosis, and angiogenesis. Recently, it was shown that breast cancer cell lines harboring a FGFR1 amplification were more sensitive to treatment with an FGFR inhibitor. To estimate if FGFR inhibitors can be a therapeutic option in human breast cancer we investigated the prevalence and the histological subtypes of FGFR1 amplification in a large cohort of breast cancers. Methods: Tissue micro-arrays with 907 breast cancers were hybridized using the commercially available fluorescent in-situ hybridization probe (FGFR1/CEN8; ZytoVision®). The results were interpreted as following: a normal gene status was considered as a ratio (FGFR1/CEN8): 0.8-1.9, an amplification was defined as a ratio β2.0, a polysomy was defined as more than >4 FGFR1 and CEP8 signals. Results: FGFR1 amplification was observed in 8.9% (n=81), a normal gene status was found in 80.7% (n=732), and a polysomy was detected in 10.2% (n=93) tumors. FGFR1 amplified breast cancers showed the following features: expression of estrogen receptor 85% (67/78), ductal 72.8% (59/81), high grade (G3) 45.5% (35/77), her2 over-expression (score2+/3+) 16% (12/75), and 18% (13/72) recurrence. Comparing T (T1, T2, T3) and N (N0 and >N0)-categories between FGFR1 amplified and non-amplified tumors the following results were seen: T1: 25% (19/77) vs. 34% (245/719), T2: 56% (43/77) vs. 48% (345/719), T3: 9% (7/77) vs. 6% (43/719), N0: 44.5% (28/63) vs 53% (338/641), >N0: 55.5% (35/63) vs 47% (303/641). We could observe a slight better long term overall survival in patients treated with anti-hormonal therapy and FGFR1 amplified breast cancers (p=0.085). Conclusions: FGFR1 amplification is prevalent (8.9%) in breast cancer, especially among the most frequent subtypes such as estrogen receptor positive breast cancers (10%) and ductal type (8%). Further, FGFR1 amplified breast cancer showed often unfavorable/aggressive features such as a high tumor grade (45.5%), larger tumor diameter, and metastasis (55.5%). These patients are a clinically relevant group since they need aggressive adjuvant treatment regimen. The detection of FGFR1 amplification could help in the identification of some patients already at higher risk which might benefit from a new therapeutic option with FGFR1 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4574. doi:1538-7445.AM2012-4574

Aims: Accurate determination of HER2 gene amplification is necessary for the selection of patients for trastuzumab therapy. FISH is regarded as the gold standard method for detecting HER2 gene amplification. In comparison to FISH, SISH has the advantages of being a method evaluated by bright-field microscopy and generating stable signals. Several studies have shown an excellent concordance between different ISH methods in detection of clearly HER2 amplified and non-amplified breast carcinomas. Aim of this study is to compare the performance of dual color SISH (dSISH) in breast carcinoma with equivocal HER2 status with conventional FISH analysis. Material&Methods: From two different Institutes, biopsies from 62 female patients with invasive breast carcinoma were investigated independently for HER2 with FISH, single color SISH (sSISH) and the dSISH technique. FDA-approved FISH method was used. HER2 gene and CEN17 signals were counted separately. The strength of linear correlation of HER2/CEN17 measurements between FISH and sSISH and between sSISH and dSISH was evaluated and agreement assessed using Bland-Altman plots, which describe the average HER2/CEN17 of the two methods versus the difference between both methods. Concordance of HER2/CEN17 assessed by FISH and dSISH for ratios of 1.5-1.8, 1.8-2.0, 2.0-2.2 and 2.2-2.5 (called categories) were determined using the weighted kappa statistic. Results: For FISH and sSISH, the correlation of HER2/CEN17 between both methods was 0.61 and considerably improved when comparing sSISH versus dSISH (correlation coefficient: 0.79). Compared to FISH, dSISH tended to over-estimate HER2/CEN17 by 0.42 signals on average, whereas 95% of all differences between FISH and dSISH ranged within a ratio of 2.4. In tumors with equivocal FISH-HER2 status (ratio 1.8-2.0 and 2.0-2.2) dSISH was highly discrepant with concordance only of 11.1% and 42.9%, respectively. In 22.2% and 28.6% of these cases dSISH results in HER2 amplification. Concordance between FISH and dSISH was greatest for HER2/C17 ratios of 1.5-1.8 and 2.2-2.5, yet kappa values still demonstrated only fair agreement (κ=0.30 and κ=0.33). Conclusion: In comparison to FISH, ISH methods like SISH have the advantage of generating stable signals and can be evaluated by bright-field microscopy. Fair agreement was achieved with dSISH when HER2/CEN17 ratio was between 1.5-1.8 and 2.2-2.5 respectively by FISH. Breast carcinoma with equivocal status for HER2 in FISH analysis was poorly reproducible with dSISH with concordance of 54% of the cases. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 334.

Abstract #5037 Background: Mastopathy is a disease with fibrocystic changes (FCC) of the breast tissue commonly seen between the ages 30-50, but rare in postmenopausal women, and therefore suggestive of an association with estrogen stimulation. Whether FCC bears an elevated breast cancer risk is unclear. The role of ESR1 amplification in FCC has not previously been investigated. Material and Methods: Paraffin embedded tissues from 59 women with invasive breast cancer (BrCa) were analyzed. For all women, tissues with FCC taken at least 1.5 years before first diagnosis of BrCa (termed here “FCC old”) were also available. Average latency between FCC and BrCa was 6years (range1.5-17y). Immunohistochemistry (IHC) with antibody against estrogen receptor alpha (ER) and FISH-analyses with a probe for ESR1 (Zytovision, Bremerhafen/Germany) were performed. As a control, tissues from 19 women with FCC without a diagnosis of in situ or invasive breast carcinoma in the observed time frame (12-18y) were taken. Clinical-pathological data, such as histological subtype, tumour stage, tumour diameter, BRE-grade, patient's age and others, were available for statistical analysis. Results: After FISH analyses, 9 of 59 (15%) BrCa were amplified for ESR1. All ESR1-amplified BrCa were strongly positive for ER with IHC (at least 80% of tumour cells labelled). Tumour type or stage and BRE-grade did not correlate with ESR1-status. In all cases of ESR1 amplification in BrCa areas of amplification in FCC adjacent to the tumour were identified. Interestingly, in women with ESR1 amplification in BrCa, an amplification detectable in tissues with FCC prior to the first diagnosis of BrCa (“FCC old”) was noted but was absent in tissues from women with FCC who did not develop BrCa in the observed time frame. Non-neoplastic ESR1 amplified cells within FCC were strongly positive for ER with IHC. In unaltered mammary glands ESR1 amplification was not observed. Discussion: In this pre-selected collective of women with BrCa and history of FCC we found amplification of the ESR1 gene in 15% of BrCa. In these women, ESR1 amplification was also detectable in tissue with FCC taken prior to the first diagnosis of BrCa. This investigation shows that ESR1 amplification is an early event in breast pathology and its occurrence in FCC predicts development of BrCa. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5037.