Genomic Characterization of Upper Tract Urothelial Carcinoma

Authors

John P. Sfakianos

Eugene K. Cha

Gopa Iyer

Sasinya N. Scott

Emily C. Zabor

Ronak H. Shah

Qinghu Ren

Aditya Bagrodia

Philip H. Kim

A. Ari Hakimi

Irina Ostrovnaya

Ricardo Ramirez

Aphrothiti J. Hanrahan

Neil B. Desai

Arony Sun

Patrizia Pinciroli

Jonathan E. Rosenberg

Guido Dalbagni

Nikolaus Schultz

Dean F. Bajorin

Victor E. Reuter

Michael F. Berger

Bernard H. Bochner

Hikmat A. Al-Ahmadie

David B. Solit

Jonathan A. Coleman

Doi

10.1016/j.eururo.2015.07.039

PMID: 26278805 · DOI: 10.1016/j.eururo.2015.07.039 · Journal: European Urology (2015)

TL;DR

Sfakianos et al. performed targeted next-generation sequencing of 300 cancer-associated genes (MSK-IMPACT) on 83 upper tract urothelial carcinoma tumors and compared the high-grade subset (n=59) to a previously sequenced cohort of 102 high-grade urothelial carcinoma of the bladder tumors. UTUC and UCB share the same gene catalog but differ in mutation prevalence: UTUC was enriched for FGFR3 (35.6% vs 21.6%), HRAS (13.6% vs 1.0%, p=0.001), and CDKN2B (15.3% vs 3.9%, p=0.016) alterations and depleted of TP53 (25.4% vs 57.8%, p<0.001) and RB1 (0% vs 18.6%, p<0.001) mutations. Activating FGFR3 mutations were found in 22 of 23 low-grade UTUC tumors, and five FGFR3-TACC3 fusions were detected exclusively in high-grade UTUC.

Cohort & data

  • 83 UTUC tumors (60 high-grade, 23 low-grade) from patients treated with radical nephroureterectomy at MSKCC, all primary urothelial histology, no predominant variants.
  • Comparator: 102 high-grade BLCA tumors without history of UTUC sequenced on the same platform (blca_mskcc_solit_2014, from ref [24] = PMID:25092538).
  • Dataset: utuc_mskcc_2015 — publicly available through MSKCC cBioPortal for Cancer Genomics.
  • Assay: MSK-IMPACT 300-gene custom hybrid-capture panel, Illumina HiSeq 2500, mean coverage 650× (UTUC) and 579× (UCB); reference genome hg19.
  • Variant calling: BWA v0.6.2 alignment, MuTect v1.0.27783 for SNVs, SomaticIndelDetector for indels, DELLY for structural rearrangements. Orthogonal validation with MiSeq and Sanger sequencing (100% concordance for 10-gene re-test).
  • Patient demographics matched between UTUC and UCB high-grade cohorts: median age 70 yr (IQR 63–76) vs 68 yr (IQR 63–76); similar sex, smoking, and stage distributions.

Key findings

  • Median 5 mutated genes per UTUC sample (n=82 after excluding one ultramutator).
  • One ultramutated tumor harbored 422 somatic mutations and a hotspot POLE V411L exonuclease-domain mutation; reported as the first POLE-driven ultramutated urothelial tumor.
  • Top 14 recurrently altered genes in UTUC included FGFR3, KDM6A, KMT2D, CDKN2A, TP53, ARID1A, TSC1, CREBBP, HRAS, PIK3CA, with 67 recurrent COSMIC missense, 31 novel missense, 114 truncating mutations, 7 amplifications, 26 homozygous deletions across the cohort.
  • 5 intrachromosomal FGFR3-TACC3 fusions identified (8.5% of UTUC vs 2.0% in UCB); breakpoints in FGFR3 intron 17 (4/5) or exon 18 (1/5) and TACC3 intron 10 (4/5) or exon 7 (1/5); confirmed by Sanger.
  • Mutually exclusive alteration pattern observed between the RTK/RAS/MAPK pathway and the p53/MDM2 pathway in UTUC (Supplementary Fig. 3).
  • High-grade UTUC vs high-grade UCB (n=59 vs n=102):
    • FGFR3 35.6% vs 21.6%, p=0.065
    • HRAS 13.6% vs 1.0%, p=0.001
    • CDKN2B 15.3% vs 3.9%, p=0.016
    • TP53 25.4% vs 57.8%, p<0.001
    • RB1 0% vs 18.6%, p<0.001
    • ARID1A 13.6% vs 27.5%, p=0.050
    • TP53/MDM2 combined: 35.6% vs 62.7%, p=0.001
    • Trends: TSC1 11.9% vs 3.9% (p=0.100); PIK3CA 10.2% vs 21.6% (p=0.084).
    • Differences (except FGFR3) remained significant after adjustment for T stage.
  • Copy-number landscape: TP53/MDM2-altered high-grade invasive UTUC (n=16) showed significantly more CNAs than FGFR3/HRAS/KRAS-mutant high-grade invasive UTUC (n=21) — rate ratios 2.91 (95% CI 1.13–7.71, p=0.028) for copy-number gains and 2.49 (95% CI 1.19–5.33, p=0.017) for total CNAs.
  • High-grade tumors carried more CNAs than low-grade (p=0.004); invasive more than noninvasive (p<0.001).
  • Low-grade UTUC: 22 of 23 (96%) harbored activating FGFR3 mutations; all 23 were TP53 and RB1 wild-type.
  • High-grade UTUC showed mutual exclusivity among FGFR3, HRAS, and TP53; TP53 mutations were found exclusively in high-grade tumors. Chromatin-modifying gene mutations were common in both low- and high-grade tumors.
  • Microdissection of 4 UTUC cases (low- vs high-grade, noninvasive vs invasive, primary vs lymph node metastasis) showed complete concordance for FGFR3 status and for KDM6A/KMT2D chromatin-modifier mutations across spatially distinct tumor components — consistent with these alterations being clonal/early events.
  • Clinicopathologic associations: TP53 (p=0.008), FGFR3 (p<0.001), CREBBP (p=0.04), KMT2C (p=0.02), and STAG2 (p=0.006) were associated with grade; TP53 with high grade, FGFR3/CREBBP/STAG2 with low grade. TP53 (p=0.002), CCND1 (p=0.046), FGFR3 (p<0.001), ERBB2 (p=0.046), ERBB3 (p=0.046), KRAS (p=0.016), and STAG2 (p=0.013) were associated with T stage.

Genes & alterations

  • FGFR3 — activating missense (e.g., S249C) in 22/23 low-grade and 27.1% of high-grade UTUC; 5 FGFR3-TACC3 gene fusions in high-grade UTUC; enriched vs UCB and associated with low grade / lower T stage.
  • HRAS — activating mutations in 13.6% of high-grade UTUC vs 1.0% of high-grade UCB (p=0.001); largely mutually exclusive with FGFR3 and TP53.
  • TP53 — 25.4% high-grade UTUC vs 57.8% high-grade UCB (p<0.001); exclusive to high-grade tumors in this UTUC cohort; associated with higher T stage and high CNA burden.
  • RB1 — 0/59 high-grade UTUC vs 18.6% high-grade UCB (p<0.001).
  • CDKN2B — homozygous deletions enriched in UTUC (15.3% vs 3.9%, p=0.016).
  • MDM2 — combined TP53/MDM2 alterations depleted in UTUC (35.6% vs 62.7%, p=0.001); MDM2-altered tumors carried high CNA burden.
  • TACC3 — partner in recurrent FGFR3-TACC3 fusions; breakpoints in intron 10 or exon 7.
  • POLE — V411L exonuclease-domain hotspot in a single ultramutator UTUC (422 somatic mutations, no focal CNAs).
  • KDM6A, KMT2D, KMT2A, KMT2C, CREBBP, EP300, ARID1A, SMARCA4 — chromatin-modifying genes; mutations common in both low- and high-grade UTUC and concordant across spatial tumor components, suggesting early/clonal events. ARID1A specifically was lower in UTUC than UCB (13.6% vs 27.5%, p=0.050).
  • TSC1 — 11.9% UTUC vs 3.9% UCB (p=0.100, trend); all mutations confirmed by orthogonal sequencing.
  • PIK3CA — 10.2% UTUC vs 21.6% UCB (p=0.084, trend).
  • CCND1, ERBB2, ERBB3, KRAS, STAG2 — significant associations with T stage in UTUC.
  • INPP4A — among the top 14 frequently altered genes in UTUC; included in orthogonal MiSeq confirmation panel.
  • CDKN2A — recurrently altered in UTUC alongside other p15/p16 pathway events.

Clinical implications

  • UTUC and UCB are biologically related but distinct: the higher prevalence of activating FGFR3 and HRAS mutations in high-grade UTUC implies a larger fraction of patients potentially eligible for FGFR or RAS/MAPK-pathway-directed therapies than the UCB experience would suggest.
  • The lower prevalence of TP53 and RB1 alterations, combined with lower CNA burden in FGFR3/HRAS/KRAS-mutant tumors, supports a “low-grade-tumor-progression” model of UTUC carcinogenesis distinct from de novo high-grade urothelial carcinogenesis dominant in UCB.
  • Authors argue routine genomic profiling of UTUC may be valuable given the high prevalence of potentially actionable alterations and the historical reliance on UCB-derived treatment paradigms.
  • Microdissection concordance for FGFR3 and chromatin-modifier mutations across primary, invasive, low-grade, and lymph-node-metastatic tumor components supports using single-region biopsies for biomarker-driven therapy selection in UTUC.

Limitations & open questions

  • 300-gene targeted panel — rare driver mutations and structural variants outside MSK-IMPACT content were not captured.
  • Epigenetic differences (promoter methylation, previously reported as more extensive in UTUC) and gene-expression differences were not analyzed.
  • High-grade UTUC enrichment for FGFR3/HRAS may partly reflect lead-time bias: difficulty of detecting and completely resecting upper-tract tumors may allow low-grade FGFR3/HRAS-mutant tumors to accumulate additional alterations and progress to high-grade disease.
  • Largest UTUC NGS series at time of publication, but authors note larger cohorts may reveal additional co-mutation patterns.
  • No survival/outcome analysis tied to mutation status is reported in this paper.

Citations from this paper used in the wiki

  • “Tumor and germline DNA from patients with UTUC (n = 83) and UCB (n = 102) were analyzed using a custom next-generation sequencing assay to identify somatic mutations and copy-number alterations in 300 cancer-associated genes.” (Abstract)
  • “Alterations more common in high-grade UTUC included fibroblast growth factor receptor 3 (FGFR3; 35.6% vs 21.6%; p = 0.065), Harvey rat sarcoma viral oncogene homolog (HRAS; 13.6% vs 1.0%; p = 0.001), and cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) (CDKN2B; 15.3% vs 3.9%; p = 0.016). Genes less frequently mutated in high-grade UTUC included tumor protein p53 (TP53; 25.4% vs 57.8%; p < 0.001), retinoblastoma 1 (RB1; 0.0% vs 18.6%; p < 0.001), and AT rich interactive domain 1A (SWI-like) (ARID1A; 13.6% vs 27.5%; p = 0.050).” (Abstract, Results)
  • “Of note, one tumor was identified to be ultramutated, harboring 422 somatic mutations and no focal copy-number alterations (CNAs)… a hotspot mutation of the exonuclease domain of polymerase (DNA directed), epsilon, catalytic subunit (POLE), V411L… To our knowledge, this is the first report of an ultramutated urothelial tumor associated with a POLE mutation.” (Section 3.1)
  • “We also identified five intrachromosomal FGFR3-transforming, acidic coiled-coil containing protein 3 (TACC3) translocations predicted to result in activating gene fusions… All five translocations were subsequently confirmed using Sanger sequencing.” (Section 3.1)
  • “TP53/MDM2-altered high-grade invasive UTUC tumors (n = 16) had significantly more copy-number gains and total CNAs (rate ratio [RR]: 2.91 [95% confidence interval (CI), 1.13–7.71; p = 0.028] vs 2.49 [95% CI, 1.19–5.33; p = 0.017], respectively).” (Section 3.2)
  • “Overall, 22 of 23 low-grade UTUC tumors were found to harbor known recurrent activating mutations in FGFR3.” (Section 3.3)
  • “There was complete concordance in FGFR3 status between the tumor components as well as the matched lymph node metastasis… Mutations in CMGs (KDM6A and KMT2D) were also concordant, supporting the hypothesis that such mutations occur early in the pathogenesis of this disease.” (Section 3.3)
  • “Genomic and associated clinicopathologic data are publicly available through the Memorial Sloan Kettering Cancer Center cBioPortal for Cancer Genomics.” (Section 2.5)

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