Sequencing urothelial carcinoma: “Just do it”

Modern medical decisions, practices, interventions and/or products can be tailored to the individual patient based on her/his predicted response or risk of disease. While the practice of customising patient treatment dates back to the time of Hippocrates, the advent of genomic-based diagnostic and informatics approaches that provide understanding of the molecular basis of disease have ushered in a new age of personalised/precision medicine.

Next-generation sequencing
This revolution was powered by advances in next-generation sequencing (NGS) technologies, which have unveiled new dimensions in cancer biology. It is now more affordable to carry out large-scale NGS experiments with a reasonable turnaround time. This has led to a rapidly expanding body of pioneering research exploring the genomic landscape and molecular mechanisms of various cancer types.

Furthermore, genetic drivers (i.e. mutations that confer a selective growth advantage, thus promoting cancer development) were discovered, exemplified by the effort from large international sequencing initiatives such as The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC). This has generated vast amounts of data and identified numerous biomarkers and targets for patient stratification and therapeutics.

Although the translation of these findings into the clinic has been slow, in certain settings (including urothelial carcinoma) NGS is becoming a complementary diagnostic tool, guiding the decision making to achieve personalised/precision medicine.

While we are still managing UC based on the “one size fits all” strategy, new NGS-based opportunities to improve our daily clinical practice are arising. In this article, we will sketch four scenarios where a tangible benefit of NGS arises for our urothelial carcinoma patients.

Sequencing for Lynch syndrome
Lynch syndrome is an autosomal dominant disorder defined by germline mutations in DNA mismatch repair (MMR) genes that predisposes individuals affected to certain malignancies. Upper tract urothelial carcinoma (UTUC) is, after colorectal and endometrial carcinoma, the third most common malignancy in Lynch syndrome¹. Reflexive testing for MMR protein loss by immunohistochemistry is, however, only recommended for colorectal and endometrial cancers. With an estimated 10-15% of UTUC cases harbouring molecular alterations common to Lynch syndrome, UTUC is the urologic disease with the highest rate of germline mutations^2,3.

Since clinical criteria are too insensitive (< 30%) to detect Lynch syndrome-related UTUC, we believe that universal screening of UTUC should be performed in all patients using NGS, to evaluate for microsatellite instability (MSI) and detect mutations of MMR genes.

Once diagnosed with Lynch Syndrome, patients have stringent protocols to diagnose other Lynch syndrome associated malignancies earlier, as they often present with earlier, aggressive tumours (especially colon cancer).

In addition, epidemiologic data and molecular characterisation suggest bladder urothelial carcinoma and prostate cancer as unrecognised components of Lynch syndrome^4,5.

Finally, Lynch syndrome related UTUCs harbour a disproportionally high rate of mutation in the tumour genome making their malignancy acutely sensitive to immune checkpoint blockade.

Sequencing for alterations in fibroblast growth factor receptors
Advanced urothelial carcinoma is a highly heterogeneous entity with a varied response to standard therapies. This multiplicity is derived from mutations, mutation signatures, chromosomal loss, and disruption of molecular pathways, which ultimately affect tumour progression, recurrence, and responsiveness to intravesical and systemic chemotherapies. The emergence of NGS has enabled a comprehensive assessment of the genomic landscape underlying advanced urothelial carcinoma, uncovering prognostic and predictive biomarkers, as well as, new therapeutic targets.

Currently we have the proof that UTUC has a distinct mutational profile compared to urothelial bladder cancer, most notably higher prevalence of fibroblast growth factor receptor (FGFR)3 alterations and a dominant APOBEC-induced mutagenesis⁶. FGFR mediates cellular proliferation and angiogenesis through activation of downstream PI3K-AKT, PKC, and Ras/MAPK pathways.

In addition, UTUC has a T-cell depleted microenvironment and a low-tumour mutational burden, all of which may contribute to a less robust antitumour immune response. If FGFR3 coordinates the luminal-papillary and immune-depleted phenotypes in UTUC, its inhibition can potentially reverse the mechanisms underlying the immune depletion. This provides a rationale for why FGFR3 inhibition may be particularly suited for the treatment of UTUC.

In advanced bladder urothelial carcinoma, FGFR activating mutations and fusions (FGFR3-TACC3) have been reported in 15-20% of patients⁷. This resulted in the current development of several agents targeting this pathway.

Erdafitinib, for example, is a pan-FGFR inhibitor that demonstrated encouraging efficacy with an objective response rate of 40% and a relatively tolerable toxicity profile in a phase II trial that includes patients with platinum-refractory metastatic bladder urothelial carcinoma harbouring FGFR2/3 mutations or fusions⁸.

Several other FGFR inhibitors are being tested in clinical trials based on FGFR mutations, fusions, and/or mRNA overexpression.

Sequencing for alterations in DNA damage response pathways
TCGA’s integrative genomic analysis of 412 patients with bladder urothelial carcinoma has demonstrated a high overall somatic tumour mutational burden (median 5.8/Mb) and the significant role of apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC) enzymes as key mutagenic drivers⁹. Somatic alterations (e.g. mutations, indels, copy number changes, fusions) were noted in several canonical pathways including TP53/cell cycle regulation (89%), RTK/RAS/PI(3)K signalling (71%), chromatin modification (52%) and DNA damage response (DDR) pathway (16%).

DDR genes sense DNA damage and promote the maintenance of genome integrity. Defects in one of the components of the DDR network lead to genomic instability, one of the hallmarks of cancer. Mutations of DDR genes such as BRCA1/2, ERCC2, ATM, FANCC or RAD51 are expected to have significant future therapeutic implications in urothelial carcinoma. For example, a mutation status of ERCC2, FANCC, ATM, and RB1 have been shown to predict response to neo-adjuvant platinum-based chemotherapies and to targeted therapies¹⁰. At the same time, DDR targeting represents an attractive therapeutic strategy especially effective in cells that already carry a DNA repair gene defect.

The paradigmatic example of DDR targeting is represented by the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib, approved as a single agent for treatment of breast and ovarian cancers harbouring BRCA1 or BRCA2 germline mutations, i.e. carrying defects in DNA repair by homologous recombination. Indeed, the presence of a loss of function mutation in a homologous recombination-related gene associated with pharmacological inhibition of a protein involved in a complementary DDR-pathway, such as PARP, leads to genomic instability and cell death.

Leveraging this putative synthetic lethality of PARP inhibition in urothelial carcinoma with deficient DDR genes has led to several trials investigating PARP inhibitors, either as monotherapy or in combination with anti-PD-1/L1 agents in advanced urothelial carcinoma.

The synergistic rationale for combining immune checkpoint with PARP inhibitors is that treatment with the latter increases the tumour mutational load, stimulates the immune recognition of the cancer cells, and upregulates PD-L1 expression.

“Further development of NGS requires real-time knowledge of genome alterations that can be used in clinical decision making”

Sequencing for molecular subtyping
Over the last decade, molecular subtyping efforts from several teams have led to distinct or partially overlapping molecular classifications of advanced bladder urothelial carcinoma. The arising molecular subtypes based on these classifications have been shown to be clinically useful in predicting the likelihood of therapy response.

Recently, a comprehensive effort of assigning 1617 muscle-invasive bladder cancer (MIBC) transcriptomes according to published independent classification systems has led to a six-class consensus system successfully reconciling the distinct structures from each input molecular classification: luminal papillary (estimated prevalence: 24%), luminal non-specified (8%), luminal unstable (15%), stroma-rich (15%), basal/squamous (35%), and neuroendocrine-like (3%)¹¹. These consensus classes differ regarding underlying oncogenic mechanisms, infiltration by immune and stromal cells, and histological and clinical characteristics, including outcomes.

This consensus system offers a robust framework that will enable testing and validation of predictive biomarkers in future prospective clinical trials. Similar as in other malignancies, such as breast cancer, molecular subtyping promises to be an integral part of pathological evaluation and drives the subsequent clinical decision-making process.

Boom in clinical trials
The advances in NGS with emerging novel therapies that target-specific key molecules in cancer cells are leading to a long-awaited boom in urothelial carcinoma translational and clinical trials. This rapid pace research is strengthening the evidence for the applicability and value of sequencing in daily practice. Indeed, NGS has been instrumental in the development of novel biomarkers, either prognostic or predictive of response to specific therapies.

However, further development of this field requires real-time knowledge of genome alterations that can be used in clinical decision making. This requires a robust data infrastructure, continuous improvement in sequencing technology, development of analytical tools based on artificial intelligence, and ongoing biomarker-driven preclinical and clinical trials.

Because of the magnitude of sequencing data generated, the continuing development of advanced bioinformatics tools capable of handling these data efficiently in a timely manner is vital for NGS-centred research and clinical implementation. Researchers and clinicians are now faced with a wide range of NGS techniques and platforms with no clear consensus guidelines, where the trade-offs between costs, accuracy, power and technical difficulties must be considered.

“NGS has the potential to guide clinicians in tailoring treatment to dynamic genomic changes in individual tumours”

Heterogeneity
Further complicating the implementation of genomic medicine is the fact that driver mutations can evolve during cancer growth. In addition to genomic evolution, tumours may also develop intertumoural and intratumour heterogeneity.

Intertumoural heterogeneity refers to differences in alterations of tumours at different sites, while intratumour heterogeneity refers to differences in alterations within a tumour. Both intertumoural and intratumour heterogeneity can further complicate the determination of relevant mutations, because it means that tissue for NGS must be obtained from relevant sites, as well as, at relevant times in the treatment course. This can result in repeated biopsies. Additionally, metastatic sites such as bone and brain can be difficult to test.

Finally, when ordering genetic tests, we must ensure that the medical, psychological, and ethical consequences of the testing have been considered. Specifically, since patient interest and awareness of genetic testing has increased with the advent of advertising campaigns¹². Moreover, we must provide accurate interpretations of tests and ensure that risk information is clearly communicated to both patient and family.

Taken together, NGS has the potential to guide clinicians in tailoring treatment to dynamic genomic changes in individual tumours, thereby tangibly improving urothelial carcinoma care beyond what we can imagine today. We are just seeing the tip of the iceberg in this genomic revolution affecting urothelial carcinoma. The question is not if sequencing will be used in daily patient care, but when and how this will happen.

The reference list can be made available to interested readers upon request by sending an email to: communications@uroweb.org.