Molecular Urothelial Tumor Cell Subtypes Remain Stable During Metastatic Evolution - Beyond The Abstract

Urothelial carcinoma (UC) is a very heterogeneous disease, especially at the molecular level, characterized by genomic instability and a high mutation rate. For this reason, different molecular subtypes have been classified in recent years, as each subtype exhibits different molecular features and clinical behaviors. As these molecular subtypes differ prognostically and subsequently show different responses to medical therapies, they are moving closer into the focus on a more tumor biology-based therapy for patients with metastatic urothelial carcinoma (mUC) to improve outcomes in the future. Consequently, subtype-stratification is currently integrated into interventional studies.

Metastatic biopsies are currently not the standard of care for treatment planning in mUC, in contrast to e.g. breast cancer to capture receptor switches during metastatic spread.¹ Of note, we have recently demonstrated that prediction of treatment response to immune checkpoint blockade is only feasible in metastatic tissue, not in matched archived primary tissue.² To date, it is not known whether the UC molecular subtypes remain stable during metastatic evolution. This is an important question prior to the widespread integration of intrinsic subtypes for treatment decisions in mUC. Therefore. we addressed this currently unresolved question of whether the molecular subtype changes during metastasis:

In the largest multicenter cohort of patient-matched syn- or metachronous metastases (MET) and primary tumors (PRIM) of patients with mUC we analyzed the stability of immunohistochemistry-based protein subtypes (well established marker panels for basal [CD44/CK5/CK14], luminal [CK20/FOXA1/GATA3/UPKII] and neuroendocrine differentiation [NCAM1, synaptophysin, chromogranin] and histomorphologies in 138 matched primary and metastatic tumor samples.³ Our study demonstrates for the first time that the protein-based tumor cell subtypes (luminal, basal, neuroendocrine; concordance 94%; 129/138 samples), as well as UC histomorphologies (concordance 94%; 130/138 samples), remain highly stable during metastatic progression. Interestingly, six PRIM tumors with conventional morphology but a small component (<10% of tumor mass) with plasmacytoid morphology appeared as pure plasmacytoid urothelial carcinoma in matched MET. This underscores the clinical relevance of even small proportions of variant/subtype histology and highlights the potential necessity to report proportions <10%.

In addition, we performed mRNA sequencing in 20 randomly selected matched pairs to investigate the stability of the transcriptome-based Consensus subtypes.⁴ The analysis revealed a lower rate of Consensus subtype concordance (45%) between PRIM and MET with the highest instability between Luminal papillary (LumP) and Luminal unstable (LumU) versus Stroma-rich subtypes even though all tumors classified as stroma-rich were luminal differentiated in protein-based tumor cell subtyping. Consistent with previous findings, Stroma-rich exhibited substantial levels of desmoplastic tumor stroma as determined from HE slides. To further investigate if luminal tumor-cell differentiation is masked by this “stromal curtain”, we developed a "luminal-ness score" based on the gene expression of KRT20, GATA3, PPARG, KRT7, FOXA1, UPK3A, UPK2, UPK1A, and assessed its correlation with stroma content in LumP, LumU, and Stroma-rich tumors. We observed a negative correlation between luminal-ness and desmoplastic stroma content concluding that tumor cell subtypes in Stroma-rich tumors are masked by the stromal tumor component and that microenvironmental factors, such as stroma-related gene expression, may influence tumor cell-related gene expression. Hence, the intrinsic tumor cell differentiation in these inconclusive cases of stroma-rich subtypes can be “rescued” by immunohistochemical subtyping.

We also explored the impact of tumor cell subtypes on the survival of patients receiving first-line platinum-based chemotherapy. Patients with luminal tumor cell subtypes exhibited prolonged overall survival, consistent with previous findings of improved responses and survival rates in luminal mUC patients undergoing platinum-based chemotherapy.^5,6 This suggests that luminal tumors may exhibit less aggressive biological behavior when treated with platinum-based chemotherapy.

Our study reveals that despite extensive clonal evolution during the progression of urothelial carcinoma, the tumor cell lineage subtype remains highly stable.^7-9 Moreover, we found that transcriptome-based subtyping provides more detailed insights into the tumor microenvironment, specifically stromal content, and can accurately capture changes in the microenvironment during metastatic evolution, which are crucial for predicting treatment outcomes. In conclusion, our findings indicate that protein-based tumor cell subtypes assessed by immunohistochemistry can be stably predicted in synchronous or subsequent metachronous metastases which have significant implications for the design of future UC subtype-stratified clinical trials.

Written by: Alexander Cox,¹ Niklas Klümper,^1,2 Markus Eckstein³ 

1. Department of Urology, University Medical Center Bonn (UKB), Bonn, Germany
2. Institute of Experimental Oncology, University Medical Center Bonn (UKB), Bonn, Germany
3. Institute of Pathology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen

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