Factors in Clonal Evolution of Chemotherapy-Resistant Urothelial Carcinoma Identified


Key Points

  • Chemotherapy-treated urothelial carcinoma is characterized by mutational heterogeneity within individual patients.
  • Chemotherapy-treated disease is enriched with clonal mutations involving L1CAM and integrin signaling pathways.

Faltas et al identified factors in the clonal evolution of chemotherapy-resistant urothelial carcinoma, according to a study reported in Nature Genetics. Findings included wide intrapatient mutational heterogeneity and enrichment for clonal mutations involving L1 cell–adhesion molecule (L1CAM) and integrin signaling pathways in chemotherapy-treated disease.

The study involved whole-exome sequencing and clonality analysis of 72 urothelial carcinoma samples, including 16 matched sets of primary and advanced tumors prospectively collected before and after chemotherapy.

"We wanted to understand how chemotherapy changes urothelial cancer and to do so we had to apply the principles of evolution," said co-first author Bishoy M. Faltas, MD, an instructor in medicine at Weill Cornell Medicine and an oncologist in the Genitourinary Oncology Program in the Division of Hematology and Medical Oncology at Weill Cornell Medicine and NewYork Presbyterian/Weill Cornell Medical Center.

"We found that chemotherapy acts as a selection pressure favoring the survival of the fittest urothelial cancer cell clones. By understanding how these urothelial cancer clones evolve at the genetic level over time and through different selective pressures such as treatment, we are hoping to translate our findings to strategies that reverse or prevent the emergence of chemotherapy resistance in bladder cancer patients.”

Major findings of the study are summarized here.

•  Chemotherapy-treated urothelial carcinoma is characterized by intrapatient mutational heterogeneity, and the majority of mutations are not shared. For example, an average of only 28.4% (range = 0.2%–76.4%) of mutations were shared in pre- and postchemotherapy samples within individual patients, with this pattern being consistent for both primary–primary tumor pairs and primary–metastatic tumor pairs. It was observed that even mutations in known driver genes, including PIK3CA, KMT2D (MLL2), ATM, and TP53, were not consistently shared in matched prechemotherapy and postchemotherapy tumors. Some postchemotherapy tumors evolved different mutations in the same key gene. The findings indicate a wide mutational heterogeneity in tumors from the same patient and an association of chemotherapy with a marked change in mutational patterns.

•  Branching evolution and metastatic spread are very early events in the natural history of urothelial carcinoma. For example, reconstruction of the evolutionary tree of one patient’s carcinoma showeda complex branching pattern. Early truncal mutations (in RYR2, ANKRD62, NCOA3, and LSS) were present in the initial founder clone and shared by all descendent clones; additional mutations were acquired at each clonal divergence node, including mutations in such driver genes as TP53 and TSC1.

By initial diagnosis, represented by tumor samples at transurethral resection of bladder tumor, at least five waves of clonal expansion from the lowest common ancestor could be identified. One of the earliest cancer cell clades had already diverged at the first divergence node and produced a cell population that metastasized to a pelvic lymph node. Subsequent removal of this lymph node at radical cystectomy may have eliminated mutations from this clade, preventing them from contributing to the development of additional metastases. However, by the time of cystectomy, additional metastasis had already occurred, derived from a different cell clade that separated at the fifth divergence node identified at diagnosis. (In this case, transition from primary to metastasis was delineated by acquisition of a non-silent mutation in the prometastatic, pro angiogenesis gene TSPAN, with this sequence suggesting a role for this mutation as a major driver in metastatic spread.)

•  Chemotherapy-treated urothelial carcinoma is enriched with clonal mutations involving L1CAM and integrin signaling pathways. There was a significant increase in the number of clonal mutations in post- vs prechemotherapy samples across the study cohort (P = .0134), confirming an association between chemotherapy and increased clonality.

Gene set enrichment analysis identified clonal enrichment of mutations in pathways involved in transmembrane transport of small molecules (odds ratio [OR] = 1.9), indicating a role for multidrug-resistance mutations in progression of advanced chemotherapy-treated disease. The analysis also identified significant enrichment in mutations in genes mediating the L1CAM (OR = 1.9) and integrin (OR = 2.8) signaling pathways; 83% and 90% of these mutations, respectively, were missense mutations that could lead to gain-of-function changes serving to activate these pathways. The findings suggest that mutations in the L1CAM and integrin signaling pathways may be involved in conferring a selective advantage for emergence of chemotherapy resistance. Further, mutations in these pathways might constitute a mechanistic link among metastatic spread, tumor microenvironment, and drug resistance in enhancing tumor survival.

•  APOBEC-induced mutagenesis is clonally enriched in chemotherapy-treated urothelial carcinoma and continues to shape disease evolution throughout its lifetime. Analysis of mutagenesis mechanisms showed signatures attributed to APOBEC mutagenesis, as well as three additional signatures attributed to age, smoking and ERCC2 mutations (the latter of which are known to be enriched in responders to cisplatin-based chemotherapy). Significant enrichment in APOBEC3-induced mutagenesis (C>T or C>G changes at TCW motifs) was observed in post- vs prechemotherapy tumors. Analysis of individual members of the APOBEC3 cytosine deaminase family showed that postchemotherapy tumors were significantly enriched for APOBEC3A-induced mutations (P = .00001) and APOBEC3B-induced mutations (P = .0395), whereas APOBEC3G mutagenesis was markedly reduced.

In addition, there was significantly increased clonality of APOBEC-induced mutations in postchemotherapy tumors; these mutations were involved in pathways important to chemotherapy resistance, including the ABC family of proteins (OR = 2.7, P = .038) and homologous recombination DNA-damage repair (OR = 3.8, P = .033). As stated by the investigators: “Our findings suggest that the APOBEC mutational process is not merely a transient event in early urothelial carcinoma oncogenesis but rather continues to shape the evolution of advanced urothelial carcinoma and may promote clonal expansions of chemotherapy-resistant clones.”

The investigators concluded: “[O]ur results demonstrate that advanced, chemotherapy-treated urothelial carcinoma undergoes extensive and dynamic clonal evolution throughout the lifetime of the tumor, with significant genetic editing that continues during and after chemotherapy. Our findings lay the foundation for an evolutionary understanding of advanced, chemotherapy-treated urothelial carcinoma and present opportunities for advancing cancer precision medicine.”

The study was supported by the Translational Research Program at Weill Cornell Medical College Pathology and Laboratory Medicine and others.

Mark A. Rubin, MD, of Weill Cornell Medicine, and Francesca Demichelis, MD, of the University of Trento and Weill Cornell Medicine, are the corresponding authors of the Nature Genetics article. Bishoy Faltas, MD, of Weill Cornell Medicine and Dr. Davide Prandi, a postdoctoral fellow at the University of Trento, are co-first authors of the paper.

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