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Molecular Characterization of Residual Bladder Cancer after Neoadjuvant Pembrolizumab

  • Andrea Necchi,
  • Joep J. de Jong,
  • Daniele Raggi,
  • Alberto Briganti,
  • Laura Marandino,
  • Andrea Gallina,
  • Marco Bandini,
  • Bashar Dabbas,
  • Elai Davicioni,
  • Umberto Capitanio,
  • Francesco Montorsi,
  • Roland Seiler,
  • Jonathan L. Wright,
  • Yair Lotan,
  • Peter C. Black,
  • Ewan A. Gibb

Background 

In patients with muscle-invasive urothelial bladder cancer (MIBC), molecular alterations in immunotherapy-resistant tumors found at radical cystectomy (RC) remain largely unstudied.

Objective 

To investigate the biology of pembrolizumab-resistant tumors in comparison to an RC cohort treated without any systemic therapy and a cohort of neoadjuvant chemotherapy (NAC)-treated tumors.

Design, setting, and participants

Transcriptome-wide expression profiling was performed on 26 RC samples from patients with ypT2-4 disease after pembrolizumab treatment, of which 22 had matched pretherapy samples. Unsupervised consensus clustering (CC) was performed to compare 26 post-pembrolizumab samples with 94 RC samples without neoadjuvant treatment and 21 samples collected from the former tumor bed of NAC-treated patients (scar tissue). Clusters were investigated for their biological and clinical characteristics and were compared to a cohort of post-NAC tumors (n = 133).

Outcome measurements and statistical analysis 

Patient and tumor characteristics were compared between subgroups using χ2 tests and two-sided Wilcoxon rank-sum tests. The primary endpoint was recurrence-free survival.

Results and limitations 

Molecular subtyping of pre- and post-pembrolizumab samples revealed significant differences: only 36% of samples had a concordant subtype according to the consensus classifier. Unsupervised CC revealed three distinct post-pembrolizumab clusters (basal, luminal, and scar-like). A scar-like subtype was present in 50% of the post-pembrolizumab cases (n = 13) and expressed genes associated with wound healing/scarring. This subtype had higher luminal marker expression in the post-pembrolizumab setting compared to CC scar-like tumors from the other cohorts. Patients with the scar-like subtype showed favorable prognosis after systemic therapy, but not in the RC-only setting. The small numbers in each subgroup represents the major study limitation.

Conclusions

This study expands our understanding of the biology of pembrolizumab-resistant MIBC and provides a framework for defining molecular subtypes after treatment. The results further support the hypothesis that luminal-type tumors may be resistant to immunotherapy or that this treatment may select for, or induce, a luminal phenotype.

Commentary by Dr. Evanguelos Xylinas

Several recent phase-2 trials have investigated the potential benefit associated with immune checkpoint inhibitors administration in the neoadjuvant setting for muscle-invasive bladder cancer (MIBC), showing rates of pathologic complete response (pCR) of 31% and 37% with 2 cycles of atezolizumab and 3 cycles of pembrolizumab, respectively. However, a range of patients would not respond to immune checkpoint inhibitors, as opposed to neoadjuvant chemotherapy. A recently published study in European Urology aimed to investigate the biology of pembrolizumab-resistant tumours in comparison with a radical cystectomy (RC) cohort treated without any systemic therapy and a cohort of neoadjuvant chemotherapy (NAC)-treated tumours.

Transcriptome-wide expression profiling was performed on 26 RC samples from patients with ypT2-4 disease after pembrolizumab treatment, of which 22 had matched pretherapy samples. Unsupervised consensus clustering was performed to compare 26 post-pembrolizumab samples with 21 samples collected from the former tumour bed of NAC-treated patients (scar tissue) and 94 RC samples without neoadjuvant treatment. Clusters were investigated for their biological and clinical characteristics and were compared to a cohort of post-NAC tumours (n = 133).

Molecular subtyping of pre- and post-pembrolizumab samples revealed significant differences: only 36% of the samples had a concordant subtype according to the consensus classifier. Unsupervised clustering revealed three distinct post-pembrolizumab clusters (basal, luminal, and scar-like). A scar-like subtype was present in 50% of the post-pembrolizumab cases (n = 13) and expressed genes associated with wound healing/scarring. This subtype had higher luminal marker expressions in the post-pembrolizumab setting compared to scar-like tumours from the other cohorts. Patients with the scar-like subtype showed favourable prognosis after systemic therapy, but not in the RC-only setting. The small numbers in each subgroup represents the major study limitation (Figure 1).

Current evidence suggests that systemic therapy significantly impacts tumour biology, characterised by molecular subtype changes after treatment. Systemic therapy-induced subtype switching was at first identified after NAC with enrichment in p53-like tumours. Then, the ABACUS study reported that 64% of the tumours had changed subtype, with enrichment of the infiltrated subtype after 2 cycles of atezolizumab administration. In the current study, 71% of post-pembrolizumab tumours were infiltrated. Noteworthily, all three studies reported a subtype characterised by fibroblast-associated markers, albeit with different nomenclatures (p53-like, scar-like, infiltrated). At present, it is unclear whether fibroblast-associated/scarring transcriptome profiles are induced by systemic treatment, whether it reflects wound healing in response to a transurethral resection of a bladder tumour (TURBT), or both.

In the PURE-01 study, residual tumours were frequently classified as luminal and lacked immune infiltration. Half (13/26) of the RC samples were classified as scar-like, but also expressed luminal markers, suggesting that luminal tumours may have an intrinsic resistance to immune checkpoint inhibitors or that pembrolizumab may select for, or induce, a luminal phenotype switch. This is consistent with the biology of luminal tumours, which have been described as having lower levels of pre-existing immune infiltration, which may be critical for a robust response to immune checkpoint therapy. Moreover, in the PURE-01 cohort, favourable pathologic responses (complete or partial) were more often seen in basal-like tumours compared to non-basal tumours.

Overall, this study expands our understanding of the biology of pembrolizumab-resistant MIBC and provides a framework for defining molecular subtypes after treatment. The results further support the hypothesis that luminal-type tumours may be resistant to immunotherapy or that this treatment may select for, or induce, a luminal phenotype. In a context in which validated molecular biomarkers are lacking, RC after neoadjuvant systemic therapy has provided an opportunity to streamline biomarker discovery through detailed molecular characterisation of the residual disease. This study provides a foundation for the development of more tailored treatments beyond immunotherapy in the future.