Case ReportA 57-year-old Caucasian woman with an unremarkable past medical history presented with lumbar pain. Physical examination revealed a mass on the right flank. An FDG-PET scan revealed a renal mass highly suggestive of malignancy, signs of liver spread, and pelvic and lumbar spinal cord bone metastases (Figure 1A). Tumor staging at initial diagnosis was pT1bN0M1 (stage IV). In April 2007, an open right radical nephrectomy was performed. The histopathologic report revealed Fuhrman grade 4 advanced ccRCC. According to the Memorial Sloan Kettering Cancer Center (MSKCC) prognostic model,14 the patient was classified as being in the intermediate-risk group based on 2 risk factors for the prognostic score: Karnofsky performance status of 70% and a serum lactic dehydrogenase level >1.5 times the upper limit of normal. She received palliative radiotherapy View Full Size Figure 1.(A) Initial tumor dissemination detected by PET/CT (arrows). (B) MRI showing skull metastasis (arrow), which was surgically removed in 2015.Citation: Journal of the National Comprehensive Cancer Network J Natl Compr Canc Netw 15, 11; 10.6004/jnccn.2017.7018Download FigureDownload figure as PowerPoint slideFigure 1.(A) Initial tumor dissemination detected by PET/CT (arrows). (B) MRI showing skull metastasis (arrow), which was surgically removed in 2015.Citation: Journal of the National Comprehensive Cancer Network J Natl Compr Canc Netw 15, 11; 10.6004/jnccn.2017.7018Download FigureDownload figure as PowerPoint slide for a painful right iliac metastasis and started sunitinib treatment at 50 mg/d on a 4 weeks on, 2 weeks off schedule (4/2). After 2 cycles, all tumoral lesions had progressed and treatment was switched to sorafenib, 400 mg twice daily. New response assessment after 2 cycles revealed disease progression with new liver and bone lesions and clinical deterioration. In March 2008, the patient started temsirolimus at 25 mg weekly and experienced a remarkable clinical benefit. She had a partial response according to RECIST criteria, with a time to best response after starting temsirolimus of 6 weeks (temsirolimus start: March 3, 2008; response at first reevaluation: April 16, 2008), and experienced overall good tolerance; she continued temsirolimus for >7 years. Main toxicity consisted of 3 different pneumonitis episodes (grade 1–3) that required dose interruptions or reductions. In August 2015, a new solitary bone metastasis arose in the skull, whereas others remained with no change (Figure 1B). A complete resection of the lesion was performed followed by local radiotherapy. At 21 months after the metastasectomy, the patient continues on treatment with biweekly temsirolimus in combination with denosumab, with no tumor progression.We performed WES in 3 different regions of the primary tumor obtained before treatment, in the skull metastasis resected 7 years after temsirolimus treatment initiation, and in the germline DNA obtained from the patient's blood. The mean depth of coverage was >80x for the 3 regions of the primary tumors, 136x for the metastasis, and 94x for the blood (see supplemental eAppendix 1 for details regarding methodology, available online with this article at JNCCN.org). The number of tumor mutations (single nucleotide variants and indels) leading to nonsynonymous coding or loss-of-function (LOF) variants in the primary tumor was 160. Tumor mutations refer to those detected in the tumor but absent in the blood. In total, 116, 76, and 65 mutations were detected in each of the 3 tumor regions and from these, 41 were shared (Figure 2; supplemental eTable 1). Among the shared tumor mutations, 39 were missense variants and 2 were LOF variants caused by frameshifts. The variants affecting genes frequently mutated in ccRCC were detected in all tumor samples analyzed: VHL (c.263G>T; p.W88L), BAP1 (c.1359dup; p.E454Rfs*15), and MTOR (c.5920T>C; p.Y1974H), and were validated by Sanger sequencing. In ccRCC the primary driver event is VHL inactivation, and thus VHL mutations are early alterations present in all tumor cells.15 The MTOR mutation frequencies detected in the different tumor samples sequenced were similar to those found for VHL, indicating that the MTOR mutation was also a truncal event in this patient (average ratio of 1.1; supplemental eTable 1). View Full Size Figure 2.Mutations revealed by whole-exome sequencing (WES). (A) Venn diagram indicating the number of tumor (absent in blood) loss-of-function and nonsynonymous coding variants identified through WES in 3 regions (R1–R3) of the primary tumor. Shared variants affecting relevant clear cell renal cell carcinoma genes are indicated below the arrow. (B) Representative genome images from the Integrative Genomics Viewer (Broad Institute), along with the number of reads for the reference (ref) and variant allele of the MTOR Y1974H mutation, and Sanger sequencing chromatogram.Citation: Journal of the National Comprehensive Cancer Network J Natl Compr Canc Netw 15, 11; 10.6004/jnccn.2017.7018Download FigureDownload figure as PowerPoint slideFigure 2.Mutations revealed by whole-exome sequencing (WES). (A) Venn diagram indicating the number of tumor (absent in blood) loss-of-function and nonsynonymous coding variants identified through WES in 3 regions (R1–R3) of the primary tumor. Shared variants affecting relevant clear cell renal cell carcinoma genes are indicated below the arrow. (B) Representative genome images from the Integrative Genomics Viewer (Broad Institute), along with the number of reads for the reference (ref) and variant allele of the MTOR Y1974H mutation, and Sanger sequencing chromatogram.Citation: Journal of the National Comprehensive Cancer Network J Natl Compr Canc Netw 15, 11; 10.6004/jnccn.2017.7018Download FigureDownload figure as PowerPoint slide View Full Size Figure 3.Functional assessment of MTOR p.Y1974H variant. Constructs expressing wild-type (WT) mTOR, mTOR Y1974H, and mTOR S2215Y (a positive control for mTOR activation) were transfected in HEK293 cells and the effect of phosphorylation of the downstream target S6K1 (pS6K1) was tested with Western blot. (A) Western blot analysis 24 hours after transfection cells were serum-starved for 24 hours (Starving) and then stimulated with 1% or 10% fetal bovine serum for 2 hours (Stimulation). In the Starving assay, lanes 1–2, 3–4, and 5–6 are duplicates from different experiments. (B) Cells transfected with mTOR constructs were treated with 2.5, 12.5, and 100 nM of rapamycin (rap) for 6 hours.Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.Citation: Journal of the National Comprehensive Cancer Network J Natl Compr Canc Netw 15, 11; 10.6004/jnccn.2017.7018Download FigureDownload figure as PowerPoint slideFigure 3.Functional assessment of MTOR p.Y1974H variant. Constructs expressing wild-type (WT) mTOR, mTOR Y1974H, and mTOR S2215Y (a positive control for mTOR activation) were transfected in HEK293 cells and the effect of phosphorylation of the downstream target S6K1 (pS6K1) was tested with Western blot. (A) Western blot analysis 24 hours after transfection cells were serum-starved for 24 hours (Starving) and then stimulated with 1% or 10% fetal bovine serum for 2 hours (Stimulation). In the Starving assay, lanes 1–2, 3–4, and 5–6 are duplicates from different experiments. (B) Cells transfected with mTOR constructs were treated with 2.5, 12.5, and 100 nM of rapamycin (rap) for 6 hours.Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.Citation: Journal of the National Comprehensive Cancer Network J Natl Compr Canc Netw 15, 11; 10.6004/jnccn.2017.7018Download FigureDownload figure as PowerPoint slide For BAP1 mutation, the average ratio was 0.66. Other genes implicated in cancer with mutations included BCL11B, CIC, EML4, KMT2C, NOTCH1, and RANBP2 (see supplemental eTable 1).To assess the effect of the MTOR novel variant p.Y1974H, HEK293 cells were transfected with constructs expressing the wild-type mTOR protein or the Y1974H variant protein. The mTOR-activating mutation S2215Y was used as control for the experiments. The Thr389 phospho-S6K1 (pS6K1) levels, which represent TORC1 activity, were augmented for the Y1974H variant compared with mTOR wild-type, and similar to those obtained for S2215Y (Figure 3A), indicating that mTOR Y1974H activates mTORC1. In addition, we observed that mTOR Y1974H was sensitive to rapamycin (Figure 3B). mTOR Y1974H also led to an increased number of cells in S phase with a concomitant decrease in G0/G1, and an alteration in forward scatter (data related to an alteration in cell size) compared with mTOR wild-type and similar to mTOR S2215Y (supplemental eFigure 1).16 Immunohistologic analysis of the tumor samples revealed activation of the mTOR pathway through a positive immunostaining of phospho-S6, a downstream target of mTOR (Figure 4A–C), with the metastasis staining being more intense than that in the primary tumor. The staining of p-ERK (Figure4 D–F) was weak in the primary tumor, whereas the metastasis exhibited a nuclear and cytoplasmatic intense and extensive staining.WES of the metastasis that arose 7 years after starting temsirolimus treatment revealed 80 tumor nonsynonymous coding or LOF variants (71 missense, 2 in-frame deletions, and 7 LOF) present in the tumor and absent in the blood. Of these, 51 were shared with the primary tumor and 29 were exclusively present in the metastasis (supplemental eTable 1). Among the genes exclusively mutated in the metastasis, only 3 (CRTC3, KAT6B, PBRM1) were in the Cancer Gene Census (CGC) but none were directly related to the MTOR pathway, and the variants have not been previously described in tumors, according to the COSMIC database.The full region of the MTOR FKBP-rapamycin binding domain (FRB), including intronic regions, was sequenced by Sanger. However, no metastasis-specific variants were detected.