Molecular classification and therapeutic targets in extrahepatic cholangiocarcinoma

Highlights

• Integrative molecular characterization of extrahepatic cholangiocarcinoma was performed.

• Targeted DNA-sequencing and whole-genome expression was conducted.

• ∼25% of extrahepatic cholangiocarcinoma have a putative actionable genomic alteration.

• Four distinct molecular classes of extrahepatic cholangiocarcinoma were identified.

Background & Aims

Cholangiocarcinoma (CCA), a deadly malignancy of the bile ducts, can be classified based on its anatomical location into either intrahepatic (iCCA) or extrahepatic (eCCA), each with different pathogenesis and clinical management. There is limited understanding of the molecular landscape of eCCA and no targeted therapy with clinical efficacy has been approved. We aimed to provide a molecular classification of eCCA and identify potential targets for molecular therapies.

Methods

An integrative genomic analysis of an international multicenter cohort of 189 eCCA cases was conducted. Genomic analysis included whole-genome expression, targeted DNA-sequencing and immunohistochemistry. Molecular findings were validated in an external set of 181 biliary tract tumors from the ICGC.

Results

KRAS (36.7%), TP53 (34.7%), ARID1A (14%) and SMAD4 (10.7%) were the most prevalent mutations, with ∼25% of tumors having a putative actionable genomic alteration according to OncoKB. Transcriptome-based unsupervised clustering helped us define 4 molecular classes of eCCA. Tumors classified within the Metabolic class (19%) showed a hepatocyte-like phenotype with activation of the transcription factor HNF4A and enrichment in gene signatures related to bile acid metabolism. The Proliferation class (23%), more common in patients with distal CCA, was characterized by enrichment of MYC targets, ERBB2 mutations/amplifications and activation of mTOR signaling. The Mesenchymal class (47%) was defined by signatures of epithelial-mesenchymal transition, aberrant TGFβ signaling and poor overall survival. Finally, tumors in the Immune class (11%) had a higher lymphocyte infiltration, overexpression of PD-1/PD-L1 and molecular features associated with a better response to immune checkpoint inhibitors.

Conclusion

An integrative molecular characterization identified distinct subclasses of eCCA. Genomic traits of each class provide the rationale for exploring patient stratification and novel therapeutic approaches.

Lay summary

Targeted therapies have not been approved for the treatment of extrahepatic cholangiocarcinoma. We performed a multi-platform molecular characterization of this tumor in a cohort of 189 patients. These analyses revealed 4 novel transcriptome-based molecular classes of extrahepatic cholangiocarcinoma and identified ∼25% of tumors with actionable genomic alterations, which has potential prognostic and therapeutic implications.

Introduction

Cholangiocarcinoma (CCA) is the second most common primary liver malignancy after hepatocellular carcinoma (HCC) and represents 1% of all neoplasms.^1,2 Although the epidemiology of CCA varies substantially worldwide, its overall incidence is increasing.³ On the basis of its anatomical origin, CCA has been classified as either intrahepatic (iCCA) or extrahepatic (eCCA), with the second-order bile ducts acting as the separation point.⁴ In addition, eCCA can be divided into perihilar (pCCA) and distal (dCCA) depending on whether they originate above or below the cystic duct.⁴ These subtypes differ not only in their location but also in their etiopathogenesis, with distinct risk factors,^3,4 different proposed cells of origin⁵ and particular genome aberrations.^6,7 Indeed, previously proposed molecular classifications of CCA (with underrepresentation of eCCA) conducted in the setting of the International Cancer Genome Consortium (ICGC)⁷ and The Cancer Genome Atlas (TCGA)⁸ initiatives have highlighted the crucial role of anatomical location in the biological underpinnings of CCA.

The therapeutic approaches for eCCA are limited. Surgical resection is the treatment of choice for early stages, although the risk of recurrence is high (>65%)⁹ leading to 5-year survival rates of around 30%. If the disease is unresectable (>65% of the cases),¹⁰ the standard of practice is gemcitabine and cisplatin, which achieves a median overall survival (OS) of 11.7 months in an unselected population of patients with biliary tract cancer (BTC).¹¹ Unfortunately, no molecular targeted therapies have demonstrated survival benefits in eCCA,¹⁰ which may be due in part to the limited understanding of the biological mechanisms driving it.

As opposed to eCCA, molecular profiling of iCCA has allowed the discovery of 2 distinct transcriptome-based classes^12,13: an “Inflammation class” with predominant induction of immune response pathways and less-aggressive clinical behavior, and a “Proliferation class” with chromosome instability and activation of classic oncogenic pathways that correlate with worse outcome. Moreover, next-generation sequencing uncovered recurrent fibroblast growth factor receptor 2 (FGFR2) translocations and isocitrate dehydrogenase 1/2 (IDH1/2) mutations in up to 20% and 15% of iCCA, respectively.^6,8,14 Early clinical trials testing FGFR inhibitors are showing promising results in patients with iCCA and FGFR aberrations.¹⁵

No integrative molecular analysis of eCCA, as a single entity, has been provided so far in Western countries. To address this, we performed a comprehensive molecular characterization of a large cohort of clinically annotated patients with eCCA (n = 189) at both genomic and transcriptomic levels. These analyses exposed major structural genomic alterations in eCCA and revealed 4 novel molecular classes with potential therapeutic implications.